Amylolytic enzyme extracted from Bacillus sp. A 7-7 (DSM 12368) and washing and cleaning agents containing this novel amylolytic enzyme

ABSTRACT

The invention relates to a novel amylolytic enzyme extracted from the microorganism  Bacillus  sp. A 7-7 (DSM 12368), to sufficiently similar proteins having an amylolytic function, to methods for the production thereof and to diverse fields of application for these proteins. In addition, the aforesaid can be further developed beyond the implemented fields of application for other technical purposes. The invention particularly relates to washing and cleaning agents containing amylolytic proteins of the aforementioned type, to methods for cleaning textiles or hard surfaces that involve the use of the amylolytic proteins or analogous agents, and to their use for cleaning textiles or hard surfaces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/590,976, filed Nov. 1, 2006, which issued as U.S. Pat. No. 7,303,905on Dec. 4, 2007, which is a divisional of U.S. application Ser. No.10/343,212, filed Jan. 28, 2003, which issued as U.S. Pat. No. 7,153,818on Dec. 26, 2006, which is the National Stage entry under 35 U.S.C. §371of International Application No. PCT/EP01/08359, filed on Jul. 19, 2001,which claims priority to German Application No. 100 36 752.2, filed onJul. 28, 2000, and German Application No. 100 36 753.4, filed on Jul.28, 2000.

SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs areincorporated herein by reference in its entirety: two copies of theSequence Listing (COPY 1 and COPY 2) on compact disc and a computerreadable form transferred pursuant to 37 C.F.R. §1.821(e) from thelast-filed computer readable form of U.S. application Ser. No.11/590,970, filed Nov. 1, 2006, each containing: file name: SequenceList-H 04713 DIV (13744-50), date recorded: Oct. 22, 2007, size: 27 KB.

This invention relates to a new amylolytic enzyme from the microorganismBacillus sp. A 7-7 (DSM 12368) and sufficiently similar proteins withamylolytic activity, to processes for their production and to variouspotential applications for these proteins. Over and above the potentialapplications mentioned, they may be further developed for other, aboveall industrial purposes. More particularly, the present inventionrelates to detergents/cleaners containing such amylolytic proteins, toprocesses for cleaning textiles or hard surfaces in which the amylolyticproteins or corresponding compositions are involved and to their use forcleaning textiles or hard surfaces.

α-Amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds ofstarch and starch-like polymers, such as amylose, amylopectin orglycogen for example, to form dextrins and β-1,6-branchedoligosaccharides. They are among the most important industrially usedenzymes. There are two reasons for this. First, like manysubstrate-degrading enzymes, they are released from microorganisms intothe surrounding medium so that they can be isolated from the culturemedium relatively easily on an industrial scale by fermentation andpurification. Second, amylases are required for a broad range ofapplications.

One important industrial use of α-amylase is the production of glucosesyrup. Other applications are, for example, as active components indetergents/cleaners, the treatment of raw materials in textilemanufacture, the production of adhesives and the production ofsugar-containing foods or food ingredients.

Enzymes such as proteases, amylases, lipases or cellulases have beenused as active components in detergents/cleaners for decades. Theirparticular contribution to the washing/cleaning performance of theparticular composition is, in the case of protease, an ability todegrade protein-containing soils, in the case of amylase the degradationof starch-containing soils and, in the case of lipase, its lipolyticactivity. Cellulases are preferably used in detergents, above all byvirtue of their contribution to the multiple wash-cycle performance of adetergent and for their fiber effect on textiles. The particularhydrolysis products are attacked, dissolved, emulsified or suspended bythe other ingredients of the detergent or, by virtue of their relativelyhigh solubility, are floated out with the wash liquor so thatsynergistic effects occur between the enzymes and the otherconstituents.

An α-amylase commonly used in detergents/cleaners is the α-amylase fromBacillus licheniformis. For example, the corresponding products of NovoNordisk A/S, Bagsvaerd, Denmark and Genencor Int., Rochester, N.Y., USAare known commercially as Termamyl® and Purastar®, respectively. Thehomolog isolated from B. subtilis or B. amyloliquefaciens and disclosedin US patent application U.S. Pat. No. 1,227,374 is marketed by NovoNordisk A/S under the name of BAN®.

This amylase molecule or its close relatives have been further developedin numerous inventions which addressed the problem of optimizing theirenzymatic properties for specific applications through variousmolecular-biological modifications. Such optimizations can relate, forexample, to the substrate specificities, to the stability of the enzymeunder various reaction conditions or to the enzymatic activity itself.The following patent applications are mentioned as examples of suchoptimizations for specific applications: EP 0 410 498 for the sizing oftextiles and WO 96/02633 for the liquefaction of starch.

Above all, however, α-amylases have been further developed in regard totheir use in detergents/cleaners. The following patent applications arementioned as just some examples of this: the amylases of WO 99/02702 aremore stable at relatively high temperatures than the starting molecule.The enzymes of WO 99/23211 are stable at high pH values, in the presenceof calcium ions and at relatively high temperatures. The α-amylases ofWO 97/43424 show a modified binding capacity for calcium ions and hencemodified enzymatic properties. The mutagenesis process of WO 99/20768leads to α-amylase variants which are particularly stable in thepresence of detergent ingredients. With modifications of the type inquestion, a change in individual enzymatic properties almost always hasan effect on other properties and on the washing performance of theparticular enzyme. One example of an optimization product obtained inthis way which is now on the market is Duramyl® (WO 94/02597) withreduced sensitivity to oxidation (Novo Nordisk A/S, Bagsvaerd, Denmark;SÖFW-Journal 123, (1997), pp. 723-731).

Since developments which merely comprise the optimization of only a fewknown starting enzymes may possibly be limited in the results obtained,there has been a parallel, intensive search for comparable enzymes fromother natural sources. This search has identified starch-splittingenzymes, for example from Pimelobacter, Pseudomonas and Thermus for foodproduction, cosmetic and pharmaceutical products (EP 0 636 693), fromRhizobium, Arthrobacter, Brevibacterium and Micrococcus (EP 0 628 630),from Pyrococcus (WO 94/19454) and Sulfolobus for starch liquefaction athigh temperatures or under highly acidic reaction conditions (EP 0 727485 and WO 96/02633). Amylases from Bacillus sp. have been found (WO95/26397 and WO 97/00324) for use at alkaline pH values. By virtue oftheir low sensitivity to detergents, other amylases from various Bacilli(EP 0 670 367) are suitable for use in detergents/cleaners.

By virtue of their origin, enzymes from newly opened organisms arepossibly more suitable than the few established enzymes for furtherdevelopment towards specific applications. One example of this is theamylase from Thermoalcalibacter (WO 98/13481) of which the naturalactivity is largely immune to calcium ions so that, from the outset, ithas the right qualifications for use in detergents.

Further optimizations of the enzymes isolated from natural sources forthe particular application can be undertaken, for example, bymolecular-biological methods (for example according to U.S. Pat. No.5,171,673 or WO 99/20768) or through chemical modifications (DE4013142). Patent application WO 99/43793, for example, describes afurther development of the known Novamyl® α-amylase. In this document,sequence similarities between Novamyl® and known cyclodextringlucanotransferases (CGTases) are used to construct a host of relatedmolecules by microbiological techniques. These related molecules areα-amylases with additional CGTase-specific consensus sequences (boxes)and functions or, conversely, CGTases with additional regions andfunctions typical of α-amylases or chimeras of both molecules. Theobject of this development is to optimize Novamyl® for theseapplications.

Patent application WO 99/57250 discloses another method for improvingthe washing performance of detergent enzymes, such as lipases,cellulases, proteases, amylases or even CGTases. The principle describedtherein consists in covalently bonding the particular enzymes tocellulose binding domains (CBDs) of bacterial origin via a non-aminoacid linker. These ensure that the enzyme acts on the surface of thetextile with greater intensity. WO 99/57252 includes other possiblelinkers in this concept while WO 99/57254 includes other enzymes suchas, for example, glycosyl transferases or acyl transferases which arebound to the CBDs either to form a chimeral protein or via the linkersmentioned in WO 99/57252.

Every amylase used for detergents has its own performance profile whichis reflected in the fact that some soils are removed more effectively byone enzyme while other soils are removed more effectively by anotherenzyme. This further demonstrates the necessity to enrich the art withother amylolytic enzymes which also have their own performance spectra.This necessity also arises from the changing habits and demands of theconsumer, according to which there is, for example, an increasing demandfor detergents for cleaning at low and medium temperatures.

In addition, new enzymes which can be obtained from organisms hithertoundeveloped for this purpose may be used as a starting product forfurther genetic engineering modifications by “protein engineering”.Their objective is to produce properties which the hitherto knownenzymes or the detergent enzymes derived from them do not or cannotpossess.

On the other hand, however, natural enzymes which, from the outset, showa certain washing or cleaning performance in conjunction with typicaldetergent ingredients seem to be particularly suitable candidates forsuch optimizations.

Despite all these developments, however, there is still a need to findother amylolytic enzymes, which a priori have a broad applicationspectrum and may be used as a starting point for specific furtherdevelopments, in addition to the few natural amylolytic enzymes whichare actually used on an industrial scale either as such or in the formof further developments.

Accordingly, the problem addressed by the present invention was toidentify a natural α-amylase hitherto undescribed which would besuitable even for industrial applications, more particularly indetergents/cleaners, or which could be used as a basis forapplication-specific further developments.

A secondary problem was to obtain the nucleic acid coding for such anα-amylase because this would be essential both for the biotechnologicalproduction and for the further development of these enzymes.

Another secondary problem was to find an organism which would naturallyproduce the particular α-amylase.

Another secondary problem was to enable the α-amylase found to bebiotechnologically produced.

Another secondary problem was to provide detergents/cleaners of whichthe washing or cleaning performance would be improved by the α-amylasefound, i.e. of which the washing or cleaning performance could be atleast partly attributed to the amylolytic protein according to theinvention.

Further secondary problems were to provide correspondingwashing/cleaning processes and to point out corresponding potentialuses.

Another secondary problem was to define further potential industrialapplications for an α-amylase which, primarily, appeared suitable foruse in detergents/cleaners.

The solution to the problem stated above and hence a first embodiment ofthe invention lies in amylolytic proteins of which the amino acidsequence is at least 96%, preferably at least 98% and more preferably100% identical with the amino acid sequence shown in SEQ ID NO. 2, moreparticularly over the region which corresponds to amino acids 32 to 516of SEQ ID NO. 2.

This includes amylolytic proteins derived from a nucleotide sequencewhich is at least 85%, preferably at least 90% and more preferably 100%identical with the nucleotide sequence shown in SEQ ID NO. 1, moreparticularly over the region which corresponds to amino acids 32 to 516of SEQ ID NO. 2. Also included are proteolytic enzymes which aresufficiently similar to these amylolytic proteins or which can bederived by methods known per se. Preferred representatives can naturallybe isolated from microorganisms, more particularly gram-positivebacteria of the genus Bacillus, especially the species Bacillus sp. A7-7 and more particularly Bacillus sp. A 7-7 (DSM 12368).

A second embodiment of the invention are nucleic acids coding foramylolytic proteins of which the nucleotide sequence is at least 85%,preferably at least 90% and more preferably 100% identical with thenucleotide sequence shown in SEQ ID NO. 1, more particularly over theregion which corresponds to amino acids 32 to 516 of SEQ ID NO. 2. Thesepreferably include—correspondingly—the nucleic acids which code for theparticular proteins of the first embodiment of the invention.

A third embodiment of the invention are the natural organisms which forma protein or derivative of the first embodiment or which contain nucleicacids coding for that protein or derivative. A particularly preferredembodiment is the strain Bacillus sp. A 7-7 which has been lodged underthe name DSM (12368).

A fourth embodiment of the invention are vectors with the nucleic acidsof the second embodiment, host cells transformed with such vectors andany biotechnological processes for the production of a protein orderivative of the first embodiment of the invention.

A fifth embodiment of the invention are detergents/cleaners which arecharacterized in that they contain a protein or derivative of the firstembodiment. These preferably include detergents/cleaners which containthe amylolytic protein or derivative in quantities of 0.000001% byweight to 5% by weight and more particularly 0.00001 to 3% by weight,which contain other enzymes, which are present in supply forms known perse or in which the amylolytic activity performs a function for therelease of the ingredients of the detergent/cleaner or is itselfcontrolled.

A sixth embodiment of the invention are processes for cleaning textilesor hard surfaces which are characterized in that an amylolytic proteinor derivative of the first embodiment becomes active in at least one ofthe process steps. Detergents/cleaners of the fifth embodiment arepreferably used for this purpose and the amylolytic protein orderivative is preferably used in a quantity of 0.01 mg to 200 mg perapplication and more particularly in a quantity of 0.02 mg to 100 mg perapplication in the particular process step.

A seventh embodiment of the invention are corresponding potentialapplications of the proteins or derivatives of the first embodiment orthe detergents/cleaners of the fifth embodiment of the invention forcleaning textiles or hard surfaces or for releasing the ingredients ofcorresponding detergents/cleaners; preferably in a quantity of 0.01 mgto 200 mg and more particularly 0.02 mg to 100 mg of the amylolyticprotein or derivative per application in a dishwasher or washingmachine.

An eighth embodiment of the invention are further potential industrialuses for the α-amylases found. These include processes for liquefyingstarch, more particularly for ethanol production, temporary bondingprocesses and various potential applications, more particularly for thetreatment of raw materials or intermediate products in textilemanufacture, more particularly for desizing cotton, for the productionof linear and/or short-chain oligosaccharides, for the hydrolysis ofcyclodextrins, for the release of low molecular weight compounds frompolysaccharide carriers or cyclodextrins, for the production of foodsand/or food ingredients, for the production of animal feeds and/oranimal feed ingredients and for dissolving starch-containing adhesivebonds.

A protein in the context of the present invention is a substantiallylinear polymer made up of the natural amino acids which generallyassumes a three-dimensional structure for performing its function. Inthe present specification, the 19 proteinogenic, naturally occurringL-amino acids are designated by the internationally accepted 1- and3-letter codes.

An enzyme in the context of the present invention is a protein whichperforms a certain biochemical function. Amylolytic proteins or enzymeswith an amylolytic function are understood to be those which hydrolyzeα-1,4-glycosidic bonds of polysaccharides, more particularly those whichlie within the polysaccharides. Accordingly, they are also referred toas α-1,4-amylases (E.C. 3.2.1.1).

Many proteins are formed as so-called preproteins, i.e. together with asignal peptide. By this is meant the N-terminal part of the protein ofwhich the function generally is to guarantee the release of the proteinformed from the producing cell into the periplasm or the surroundingmedium and/or its correct folding. The signal peptide is then split offfrom the rest of the protein under natural conditions by a signalpeptidase so that it performs its actual catalytic activity without theN-terminus initially present. The native α-amylase from Bacillus sp.A7-7 (DSM 12368), for example, is 516 amino acids long, as shown in SEQID NO. 2. As shown in SEQ ID NO. 1, the signal peptide of this enzymecomprises 31 amino acids so that the mature enzyme has a length of 485amino acids.

By virtue of their enzymatic activity, the mature peptides, i.e. theenzymes processed after their production, are preferred to thepreproteins for industrial applications.

Proproteins are inactive precursors of proteins. Their precursors withsignal frequency are known as pre-proproteins.

In the context of the present invention, nucleic acids are the moleculesnaturally made up of nucleotides which serve as information carriers andwhich code for the linear amino acid sequence in proteins or enzymes.They may be present as a single strand, as a single strand complementaryto that single strand or as a double strand. As the naturally morepermanent information carrier, the nucleic acid DNA is preferred formolecular biological work. By contrast, for carrying out the inventionin a natural environment, for example in an expressing cell, an RNA isformed so that RNA molecules essential to the invention also representembodiments of the invention.

With DNA, the sequences of both complementary strands in all threereading frames have to be considered. Another factor to be considered isthat different codon triplets can code for the same amino acids, so thata certain amino acid sequence can be derived from several differentnucleotide sequences possibly having only minimal identity(degenerateness of the genetic code). In addition, different organismsshow differences in the use of this codon. For these reasons, both aminoacid sequences and nucleotide sequences have to be included in theconsideration of the scope of protection and disclosed nucleotidesequences should only be regarded as an exemplary coding for a certainamino acid sequence.

The unit of information corresponding to a protein is also referred toas a gene in the present specification.

With the help of methods now generally known, such as for examplechemical synthesis or the polymerase chain reaction (PCR), inconjunction with molecular-biological and/or protein-chemical standardmethods, the expert is able to produce the corresponding nucleic acidsup to and including complete genes on the basis of known DNA and/oramino acid sequences. Such methods are known, for example, from the“Lexikon der Biochemie”, Spektrum Akademischer Verlag, Berlin, 1999,Vol. 1, pp. 267-271 and Vol. 2., pp. 227-229.

Changes in the nucleotide sequence, which can be produced for example bymolecular-biological methods known per se, are referred to as mutations.Depending on the type of change, mutations are known, for example, asdeletion, insertion or substitution mutations or mutations where variousgenes or parts of genes are fused together (“shuffling”); these are genemutations. The associated organisms are known as mutants. The proteinsderived from mutated nucleic acids are referred to as variants. Forexample, deletion, insertion or substitution mutations or fusions leadto deletion-, insertion-, substitution-mutated or fusion genes and, atthe protein level, to corresponding deletion, insertion or substitutionvariants or fusion proteins.

Fragments are understood to be any proteins or peptides which aresmaller than natural proteins or those which correspond to completelytranslated genes and which, for example, can also be syntheticallyobtained. On the basis of their amino acid sequences, they can beassigned to the particular complete proteins. For example, they mayassume identical structures or may perform proteolytic activities orpartial activities such as, for example, the complexing of a substrate.Fragments and deletion variants of starting proteins are basically thesame. Whereas fragments are relatively small pieces, deletion mutantslack only short regions and hence only individual partial functions.

In the context of the present invention, chimeral or hybrid proteins areproteins made up of elements which naturally emanate from differentpolypeptide chains from the same organism or from different organisms.This procedure is also known as shuffling or fusion mutagenesis. Theobject of such a fusion can be, for example, to produce or modify acertain enzymatic function with the aid of the fused-on part of theprotein.

Proteins obtained by insertion mutation are understood to be variantswhich have been obtained by methods known per se by insertion of anucleic acid or protein fragment into the starting sequences. Becausethey are basically the same, they may be assigned to the chimeralproteins from which they differ solely in the size ratio of theunchanged part of the protein to the size of the entire protein. Ininsertion-mutated proteins, the proportion of foreign protein is lowerthan in chimeral proteins.

Inversion mutagenesis, i.e. partial sequence inversion, may be regardedas a special form of both deletion and insertion. The same applies to aregrouping of various parts of the molecule which differs from theoriginal amino acid sequence. They may be regarded as a deletionvariant, as an insertion variant and as a shuffling variant of theoriginal protein.

Derivatives in the context of the present invention are proteins ofwhich the pure amino acid chain has been chemically modified. Suchderivatizations can be carried out, for example, biologically inconnection with protein biosynthesis by the host organism.Molecular-biological methods may be used for this purpose. They may alsobe carried out chemically, for example by the chemical conversion of aside chain of an amino acid or by covalent bonding of another compoundto the protein. This compound may be, for example, another protein whichis bound to proteins according to the invention, for example bybifunctional chemical compounds. Derivatization is also understood toinclude covalent bonding to a macromolecular carrier.

In the context of the invention, all enzymes, proteins, fragments andderivatives come under the collective heading of proteins unless theyneed to be explicitly referred to as such.

Vectors in the context of the invention are understood to be elementsconsisting of nucleic acids which contain an interesting gene as acharacteristic nucleic acid region. They are able to establish this in aspecies or a cell line over several generations or cell divisions as astable genetic element which replicates independently of the rest of thegenome. Vectors are special plasmids, i.e. circular genetic elements,particularly where they are used in bacteria. In genetic engineering, adistinction is drawn between, on the one hand, vectors which are usedfor storage hence also for genetic work so to speak (the so-calledcloning vectors) and, on the other hand, vectors which perform thefunction of producing the interesting gene in the host cell, i.e.facilitating the expression of the particular protein. These vectors areknown as expression vectors.

By comparison with known enzymes, which are lodged for example ingenerally accessible data banks, characteristic molecule parts such asstructural elements, for example, or the enzymatic activity of a studiedenzyme can be deduced from the amino acid or nucleotide sequence. Such acomparison is made by assigning similar sequences in the nucleotide oramino acid sequences of the studied proteins to one another. This isknown as homologizing. A tabular assignment of the particular positionsis known as alignment. In the analysis of nucleotide sequences, bothcomplementary strands and all three possible reading frames have to betaken into consideration, as do the degenerateness of the genetic codeand the organism-specific codon usage. Alignments are now produced bycomputer programs, for example by the FASTA or BLAST algorithms; thisprocedure is described, for example, by D. J. Lipman and W. R. Pearson(1985) in Science, Vol. 227, pp. 1435-1441. A compilation of allpositions in accord in the compared sequences is known as a consensussequence.

Such a comparison also provides information on the similarity orhomology of the compared sequences to one another. This is expressed inpercent identity, i.e. the proportion of identical nucleotides or aminoacid residues at the same positions. A more broadly defined notion ofhomology includes the conserved amino acid exchanges in this value.Percent identity then becomes percent similarity. Such assertions can bemade about whole proteins or genes or only about individual regions.

Homologous regions of different proteins are generally those with thesame structural elements and/or functions which can be recognized byaccordances in the primary amino acid sequence. It extends to completeidentities in very small regions, so-called boxes, which comprise only afew amino acids and which generally perform essential functions for theoverall activity. By functions of the homologous regions are meant verysmall partial functions of the function performed by the protein as awhole, such as for example the formation of individual hydrogen bridgebonds for complexing a substrate or transition complex.

The enzymatic activity can be qualitatively or quantitatively modifiedby other regions of the protein which do not take part in the actualreaction. This concerns, for example, the enzyme stability, activity,reaction conditions or substrate specificity.

Accordingly, the definition of an amylolytic protein according to theinvention does not apply just to one with the pure function of carryingout the hydrolysis of a α-1,4-glycosidic bonds which are attributable tothe few amino acid residues of a probable catalytically active center.It also encompasses all the functions supporting the hydrolysis of anα-1,4-glycosidic bond. Such functions can be performed, for example, byindividual peptides and by one or more individual parts of a protein byacting on the actual catalytically active regions. The definition of theamylolytic function also encompasses such modifying functions alone.This is because, on the one hand, it is not known exactly which aminoacid residues of the protein according to the invention actuallycatalyze the hydrolysis and, on the other hand, certain individualfunctions cannot be definitively excluded from the outset fromparticipation in the catalysis. The auxiliary functions or partialactivities include, for example, the binding of a substrate, anintermediate or end product, the activation or the inhibition orimparting of a controlling effect on the hydrolytic activity. This canalso involve, for example, the formation of a structural element whichlies far from the active center or a signal peptide of which thefunction concerns the release of the protein formed from the cell and/orits correct folding and without which no enzyme capable of functioningis generally formed in vivo. Overall, however, α-1,4-glycosidic bonds ofstarch or starch-like polymers must be hydrolyzed.

The performance of an enzyme is understood to be its effectiveness inthe technical field under consideration. This is based on the actualenzymatic activity, but is also dependent on other factors relevant tothe particular process. These include, for example, stability, substratebinding, interaction with the material carrying the substrate orinteractions with other ingredients, more particularly synergisms. Forexample, consideration of whether an enzyme is suitable for use indetergents will also include an assessment of its contribution to thewashing or cleaning performance of a detergent or cleaner formulatedwith other constituents. An enzyme can be further developed andoptimized for various technical applications using molecular-biologicaltechniques known per se, more particularly those mentioned in theforegoing.

Under the Budapest Treaty over the international recognition of thelodging of microorganisms of 28 Apr. 1977, Bacillus sp. A 7-7 wasdeposited for the present invention on 5 Aug. 1998 in the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (German Collection ofMicroorganisms and Cell Cultures GmbH), Inhoffenstralβe 7 B, 38124Braunschweig, Germany (DSMZ). The deposited Bacillus sp. A 7-7 carriesthe registration number DSM 12368 (DSM 98-587). The deposit was made inaccordance with the terms and provisions of the Budapest Treaty relatingto deposit of microorganisms and was made for a term of at least thirty(30) years and at least (5) years after the most recent request for thefurnishing of a sample of the deposit is received by the depository, orfor the enforceable life of the patent, whichever is longer, and will bereplaced if it becomes non-viable during that period. The key datarelating to the features of this biological material, as determined bythe DSMZ on the lodgement date, are set out in Table 1 below.

TABLE 1 Microbiological properties of Bacillus sp. A 7-7 (DSM 12368) (asdetermined by the DSMZ on the Sep. 10, 1998) Property Result Cell formRodlets width [μm] 3.0-4.5 length [μm] 0.8-1.0 Spores Positive/ovalSporangium Slight swollen Oxidase Positive Catalase Positive Anaerobicgrowth Positive VP reaction Negative pH in VP medium 9.1 Growth at 40°C. Positive/weak Growth at 50° C. Negative Growth in medium pH 7.0Negative NaCl 2% Positive NaCl 5% Positive NaCl 7% Positive NaCl 10%Positive NaCl 12% Negative NaCl 16% Negative lysozyme medium PositiveAcid from D-glucose Negative L-arabinose Negative D-xylose NegativeD-mannitol Positive D-fructose Positive Hydrolysis of starch Positivegelatin Positive casein Positive tyrosine Weak Tween 80 Positive Tween60 Positive Tween 40 Positive Tween 20 Negative Lecithinase PositivePullulan Positive Hydrolysis of hippurate Positive Esculin PositiveUtilization of citrate Positive Propionate Positive NO₂ from NO₃Positive Indole reaction Negative Phenyl alanine desaminase NegativeRESULT Bacillus sp. (RNA group VI, alcaliphilic) Remarks Thephysiological test results point to the species B. alcalophilus or B.horikoshii, but cannot clearly identify any of the species mentioned.The strain showed 2 colony forms which were determined as variants ofone and the same species by fatty acid analysis. Partial sequencing ofthe 16S rDNA produced 94.8% accordance with B. alcalophilus. Strain A7-7 is probably the representative of a new species.

Now, as has been surprisingly found over and above thischaracterization, the amylolytic enzyme produced by this strain hasproperties which predestine it for use in a number of industrialprocesses. In addition, the strain has properties which favorably affectcultivatability.

As shown in detail in Example 2, the amylolytic enzyme according to theinvention of the strain Bacillus sp. A 7-7 (DSM 12368) may bebiochemically characterized as follows: as a mature protein, it has anapparent molecular weight of 58 kD in denaturing SDS polyacrylate gelelectrophoresis whereas a molecular weight of around 59 kD can bederived from the protein sequence of 516 amino acids (SEQ ID NO. 2) andone of 55.5 kD after removal of the signal peptide comprising 31 aminoacids. According to isoelectric focussing, the isoelectric point of themature protein is 6.0. It has amylolytic activity. It is stable toincubation for 10 mins. at pH 10/50° C. 50% residual activity isobserved at 60° C. The enzyme is largely stable to incubation for 10minutes at 40° C./pH 5-12, the best stability being observed at pH 9. Inthe presence of 0.1% SDS, the enzyme shows 98% residual activity afterincubation for 15 minutes at pH 10/50° C. In the presence of anadditional 10 HPE/ml protease activity and after incubation for 15 mins.at pH 10/50° C., the enzyme still has 74% residual activity.

Accordingly, the present invention provides a naturally occurring enzymewhich must be regarded as α-amylase on the strength of its sequencehomologies to the hitherto known enzymes and its enzymatic activity. Inprinciple, it may be used for any applications which require anamylolytic function. It is particularly suitable for applicationsinvolving alkaline pH values and medium temperature ranges, moreparticularly pH values above 9 and/or temperatures above 40° C. Theapplication spectrum is extended by the comparatively high stability ofthe enzyme to detergents and proteases. Accordingly, it appears to beparticularly suitable for use in detergents/cleaners.

The nucleotide sequence of this enzyme is shown in the sequence protocolunder the heading SEQ ID NO. 1. Accordingly, it is available, forexample, for further developments using molecular-biological methodsknown per se. The amino acid sequence of the enzyme is shown in thesequence protocol under the heading SEQ ID NO. 2.

Comparable amylolytic proteins are also embodiments of the presentinvention and are claimed insofar as they have protein or DNA sequenceswhich lie within the range of similarity to the sequences shown in SEQID NO. 1 and/or SEQ ID NO. 2. This similarity range encompasses allproteins of which the amino acid sequence is at least 96%, 96.5%, 97%,97.5%, 98%, 98.5%, 99%, 99.5% or 100% identical with the amino acidsequence shown in SEQ ID NO. 2. The similarity range also encompassesall proteins of which the nucleotide sequence is at least 85%, 87.5%,90%, 92.5%, 95%, 96%, 97%, 98%, 99% or 100% identical with thenucleotide sequence shown in SEQ ID NO. 1. This applies in particular tothose partial ranges of the protein which relate to amino acids 32 to516.

The nearest similar protein known on Mar. 17, 2000 is the α-amylase fromBacillus alcalophilus with the registration number P 19571 in theSwiss-Prot data bank (Geneva Bioinformatics (GeneBio) S.A., Geneva,Switzerland). This protein has a sequence homology of 93.4% identity atprotein level to the amylolytic enzyme according to the invention fromBacillus sp. A 7-7 (DSM 12368). The protein according to the inventionis clearly characterized as α-amylase through the homologous regions.Representative related proteins are presented in the alignment in FIG.1.

On the basis of this alignment, largely the same secondary and tertiarystructures may be assumed for proteins according to the invention as forthe proteins used for homologizing. Their structural elements may beretrieved from generally accessible data banks such as, for example, theEMBL European Bioinformatics Institute (EBI) in Cambridge, UK,Swiss-Prot or GenBank (National Center for Biotechnology InformationNCBI, National Institutes of Health, Bethesda, Md., USA). If differingstructures should appear or if it should turn out that there are variousfolding variants with varying amylolytic properties, for example so faras the optimal reaction conditions or the substrate specificity isconcerned, these are all included in the scope of protection of thepresent invention. This is because, firstly, the folding can depend uponthe production conditions, for example in the presence or absence of theleader peptide. Secondly, these variants can turn out to be particularlysuitable for various potential applications, for example for thequantitative liquefaction of starch, for the hydrolysis of cyclodextrinsor for use in detergents/cleaners.

Particular interest attaches to the partial sequence corresponding toamino acids 32 to 516 from the sequence shown in SEQ ID NO. 2. This isbecause, as can be concluded from the amino acid sequence, the first 31amino acids represent a signal peptide which, in the case of productionin corresponding microorganisms, probably initiates the release of theprotein from the cell interior into the medium surrounding the cells.After the release, this signal peptide is split off in vivo so that theactual amylolytic activity is developed by the remaining part of theprotein.

Accordingly, for the actual amylolytic function, amino acids 1 to 31 areprobably of little significance, but are of importance for productionand particularly for the required folding. Because of this, they cannotbe excluded from the scope of protection of the present invention.

Should it turn out that there are deviations in the length of the signalpeptide and/or the mature protein during production, for example by oneor other bacterial strain, the claims relating to positions 1 to 31 or32 to 516 according to SEQ ID NO. 2 apply accordingly to thecorresponding variants. For example, the transition of the protein fromBacillus sp. A 7-7 (DSM 12368) could use as many as six nucleotidesbefore the nucleotide sequence shown in SEQ ID NO. 1. Before thisprobable beginning lie the six nucleotides ATG ACG. These could betranslated as Met-Thr so that the signal peptide is N-terminallylengthened by two amino acids and a certain similarity to the amylasefrom Bacillus sp. # 707 shown in FIG. 1 under number 2 (SEQ ID NO: 3) isobtained. The splitting off the signal peptide also lends itself tovariation.

Of the variants falling within the similarity range mentioned above,those which have optimized properties for the potential applicationsenvisaged are particularly preferred. As explained at the beginning,such variants can be produced by methods, preferablymolecular-biological methods, known per se. For example, it would alsobe possible to delete methionine, tryptophane, cysteine and/or tyrosineresidues of proteins according to the invention and/or to replace themwith less readily oxidizable amino acid residues in accordance with theteaching of WO 94/18314. Oxidation stability, the pH activity profileand/or thermal stability can be improved in this way. Furtherdevelopments through point mutagenesis may also be carried, for example,in accordance with WO 99/09183 and WO 99/23211.

Fragments according to the invention are understood to be any proteinsor peptides that are smaller than the proteins which correspond to thoseof SEQ ID NO. 1 or SEQ ID NO. 2, but are sufficiently homologous to themin the corresponding partial sequences. If they develop an amylolyticfunction or at least a function that supports the hydrolysis of anα-1,4-glycosidic bond, they are regarded as amylolytically activefragments and represent embodiments of the present invention. Thisapplies, for example, to fragments which contribute to the complexing ofa substrate or to the formation of a structural element necessary forthe hydrolysis. The fragments may be, for example, individual domains orfragments which do not accord with the domains. Such fragments can beproduced relatively inexpensively, no longer have certain possiblyunfavorable characteristics of the starting molecule, such as possiblyan activity-reducing regulating mechanism, or may develop a morefavorable activity profile. Such protein fragments can also be produced,for example, chemically rather than biosynthetically. Chemical synthesiscan be advantageous, for example, when chemical modifications are to bemade after the synthesis.

By virtue of their basic similarity, proteins obtainable by deletionmutation may also be assigned to the fragments. Such proteins maylargely correspond biochemically to the starting molecules or no longerhave individual functions. This appears particularly appropriate, forexample, in the deletion of inhibiting regions. In the final analysis,the deletions may be used both for specialization and for extending therange of application of the protein. If an amylolytic function in thebroadest sense is maintained, modified, specified or even achieved inthe first place in this way, the deletion variants and the fragments areproteins according to the invention. The only additional requirement inthis regard is that—over and above the homologous partial sequence stillpresent—they should lie within the above-mentioned similarity range tothe sequences SEQ ID NO. 1 and SEQ ID NO. 2.

For example, it is possible in accordance with WO 99/57250 to provide aprotein according to the invention or parts thereof with binding domainsfrom other proteins via peptidic or nonpeptidic linkers and thus to makehydrolysis of the substrate more effective. Such constructs fall withinthe scope of protection of the present invention when they developamylolytic activities and those parts of the construct which performthis function are sufficiently similar to the stated sequences accordingto the invention. Equally, amylolytic proteins according to theinvention may also be linked, for example, to proteases in order toperform a double function.

The proteins and signal peptides obtainable from preproteins bysplitting off the N-terminal amino acids may also be regarded asnaturally formed fragments or deletion-mutated proteins. A splittingmechanism such as this may also be used to predetermine specificcleavage sites in recombinant proteins with the aid of certain sequenceregions that are recognized by signal peptidases. Proteins according tothe invention can thus be activated and/or deactivated in vitro. Thescope of protection of the present invention encompasses each of theseproteins providing it falls within the claimed scope of protection andimparts amylolytic activity.

Chimeral or hybrid proteins according to the invention are understood tobe proteins which are made up of elements emanating naturally fromvarious polypeptide chains. This procedure is also known as shuffling orfusion mutagenesis. Proteins are chimeral proteins according to theinvention when the proteins obtained by fusion have amylolytic activityin the broadest sense. This may be developed or modified by a part ofthe molecule which derives from a protein according to the invention andlies within the claimed similarity range. The object of such a fusioncan be, for example, to produce or modify an amylolytic function or afunction supporting the hydrolysis of α-1,4-glycosidic bonds with theaid of the fused-on part of the protein according to the invention. Inthe context of the invention, it does not matter whether such a chimeralprotein consists of a single polypeptide chain or of several subunitsamong which various functions can be distributed. In order to realizethe second alternative, it is possible, for example, to split a singlechimeral polypeptide chain into several by controlled proteolyticcleavage either post-translationally or after a purification step. Thepresent invention also relates to chimeral proteins which, by virtue oftheir construction, have an optionally lower identity over their entireamino acid and/or nucleotide sequence than defined above for thesimilarity range according to the invention, but may be assigned to itin at least one of the regions introduced by fusion and perform the samefunctions in this part as in an amylase which falls within theabove-mentioned homology range over its entire length.

Proteins according to the invention obtainable by insertion mutation arevariants of the proteins which fall over their entire sequence lengthinto the designated range of protection of the SEQ ID NO. 1 or SEQ IDNO. 2 sequences and which have been obtained by insertion of a nucleicacid or protein fragment into the respective sequences. As with hybridformation, the object of insertion mutagenesis can be to combineindividual properties of proteins according to the invention with thoseof other proteins. Proteins are proteins according to the inventionobtained by insertion mutation or chimeral proteins when the regions tobe attributed through their homology to the SEQ ID NO. 1 or SEQ ID NO. 2sequences have corresponding homology values and the protein has anamylolytic function in the broadest sense by virtue of those regions.

Accordingly, proteins obtained by inversion mutagenesis and those with aregrouping of various parts of the molecule which differs from theoriginal amino acid sequence are included in the scope of protection ofthe present invention. It may be regarded as a deletion variant, aninsertion variant or as a shuffling variant of the original protein.

Amylolytically active derivatives according to the invention areunderstood to be amylolytic proteins which have been modified, forexample in connection with protein biosynthesis through processing bythe host organism or chemically, for example by the transformation of aside chain of an amino acid or by covalent bonding of another compoundto the protein. This compound may consist, for example, of otherproteins which are bound to proteins according to the invention, forexample by bifunctional chemical compounds. Such modifications caninfluence, for example, the substrate specificity or the strength of thebond to the substrate or can temporarily block the enzymatic activitywhere the coupled substance is an inhibitor. This may be appropriate,for example, for the duration of storage. Another embodiment arederivatives which have been obtained by covalent bonding to amacromolecular carrier such as, for example, polyethylene glycol or apolysaccharide.

Other solutions to the problem addressed by the invention are amylolyticproteins or derivatives which have at least one antigenic determinant incommon with one of the above-mentioned proteins or derivatives.

This is because the development of enzymatic activities is criticallydetermined not just by the pure amino acid sequence of a protein, butalso by its secondary structural elements and its three-dimensionalfolding. Thus, domains differing clearly from one another in theirprimary structure can form spatially substantially correspondingstructures and can thus provide for the same enzymatic behavior. Suchcommon features in the secondary structure are normally recognized ascorresponding antigenic determinants of antisera or pure or monoclonalantibodies. Structurally similar proteins or derivatives can thereforebe detected and assigned through immunochemical cross reactions.

Accordingly, the scope of protection of the present invention alsoencompasses proteins or derivatives which have amylolytic activity andwhich can possibly be assigned to the above-defined proteins accordingto the invention or derivatives through their immunochemicalrelationship, but not through their homology values in the primarystructure.

Proteins according to the invention which emanate from natural sourcesare preferred embodiments of the present invention, particularly wherethey originate from such microorganisms as single-cell fungi orbacteria. This is because such microorganisms are easier to handle thanmulticell organisms or cell cultures derived from them. These representappropriate options for special embodiments.

Proteins or derivatives according to the invention from gram-positivebacteria are particularly preferred because they do not have an externalmembrane and therefore directly release secreted proteins into thesurrounding medium.

Proteins or derivatives according to the invention from gram-positivebacteria of the genus Bacillus are most particularly preferred becausethey are established as production organisms with a particularly highproduction performance in industrial processes.

Of the proteins or derivatives according to the invention from Bacillusspecies, those from alcaliphilic bacilli are preferred, those fromBacillus sp. A 7-7 being particularly preferred and those from thestrain Bacillus sp. A 7-7 (DSM 12368) most particularly preferred. Thisis because the embodiment of the enzyme according to the invention ofwhich the associated sequences are shown in the sequence protocol and ofwhich the enzymatic characteristics are described in the Examples wasoriginally obtained from that strain.

Strains which release the amylolytic protein formed into the mediumsurrounding them are preferred for production reasons.

It is possible that, although naturally occurring producers can producean amylolytic enzyme according to the invention, they only express itand/or release it into the surrounding medium to a minimal extent underthe conditions initially determined. They still fall within the scope ofprotection of the present invention as long as it is possibleexperimentally to determine suitable environmental conditions or lowmolecular weight or other factors under whose influence they can bestimulated to produce the protein according to the invention on a levelwhich makes economic utilization appear appropriate. A regulatingmechanism such as this can be purposefully used for biotechnologicalproduction, for example for regulating the responsible promoters.

Depending on its isolation, working up or preparation, a protein can beassociated with various other substances, particularly if it has beenrecovered from natural producers of the protein. Certain othersubstances may then—or even independently—have been purposefully addedto it, for example to increase its stability in storage. Accordingly,the definition of the protein according to the invention alsoencompasses all preparations of the actual protein essential to theinvention. This is also independent of whether or not it actuallydevelops this enzymatic activity in a certain preparation because it canbe desirable for the protein to have little or no activity in storageand only to develop its amylolytic activity at the time of use. This candepend, for example, on the folding status of the protein or can resultfrom the reversible binding of one or more companion substances from thepreparation or from another control mechanism.

Proteins according to the invention, particularly in storage, can beprotected by stabilizers, for example against denaturing, disintegrationor inactivation, for example by physical influences, oxidation orproteolytic cleavage. In the case of proteins obtained frommicroorganisms, inhibition of proteolysis is particularly criticalbecause most microorganisms secrete various proteases as digestiveenzymes into the surrounding media. Such enzymes can seriously damagethe interesting proteins during the subsequent purification steps.

One group of stabilizers are reversible protease inhibitors such as, forexample, benzamidine hydrochloride and leupeptin, borax, boric acids,boron acids, salts or esters thereof, peptide aldehydes or pure peptidicinhibitors, such as ovomucoid or specific subtilisin inhibitors. Othercommon enzyme stabilizers are aminoalcohols, such as mono-, di-,tri-ethanolamine and -propanolamine, aliphatic carboxylic acids up toC₁₂, dicarboxylic acids, lower aliphatic alcohols, but above all polyolssuch as, for example, glycerol, ethylene glycol, propylene glycol orsorbitol. Calcium salts such as, for example, calcium acetate or calciumformate, magnesium salts, various polymers, such as for example lignin,cellulose ethers, polyamides or water-soluble vinyl copolymers, are alsoused to stabilize the enzyme preparation, above all against physicalinfluences or pH variations. Reducing agents and antioxidants, such assodium sulfite or reducing sugars for example, increase the stability ofthe proteins against oxidative disintegration.

The present invention is also embodied in corresponding nucleic acidsproviding the nucleic acids in question code for an amylolytic proteinin the broadest sense and show sufficient similarity—as defined above—tothe SEQ ID NO. 1 sequence, more particularly in nucleic acids which codefor a protein that corresponds to the partial range of amino acids 32 to516 of the amino acid sequence shown in SEQ ID NO. 1.

Particularly preferred embodiments are nucleic acids which code for oneof the above-described amylolytic proteins according to the invention.This also includes variants which do not fall within the similarityrange defined in SEQ ID NO. 1 over their entire sequence length, but doso in individual regions. These include, for example, the nucleotidesequences which, as explained above, have been obtained by insertion ordeletion mutation, chimeral proteins or protein fragments. However,so-called antisense constructs, for example through individual partialsections, also represent embodiments of the present invention becausethey can be used to regulate the amylolytic activity.

Nucleic acids form the starting point for molecular-biologicalinvestigations and further developments. Such methods are described, forexample, in the manual by Fritsch, Sambrook and Maniatis “Molecularcloning: a laboratory manual”, Cold Spring Harbour Laboratory Press, NewYork, 1989. All the genetic engineering and protein-biochemical methodswhich come under the heading of protein engineering in the prior art arealso based on the gene, particularly the cloned gene. Proteins accordingto the invention can be further optimized for various uses by suchmethods, for example by point mutagenesis or by fusion with sequencesfrom other genes.

The variants according to the invention of a protein obtainable bymolecular-biological methods known per se include in particular thosewith individual, specific amino acid exchanges or randomized pointmutations, deletions of individual amino acids or of partial sequences,fusions with other fragments or other enzymes, insertions or inversions,i.e. partial sequence inversions. Such mutations or modifications canrepresent preferred embodiments for specific applications. Such amutagenesis can be carried out purposefully or by random methods. It canbe combined, for example, with a subsequent activity-directed screeningand selection process on the cloned genes. The genes obtained bymutation fall within the scope of protection of the present inventionproviding they code for amylolytic proteins in the broadest sense andfall within the similarity range defined above, at least in thehomologous and functionally relevant regions.

Another solution to the problem addressed by the invention and henceanother embodiment of the invention are the organisms which naturallyform a protein according to the invention or derivative or containnucleic acids which code for a protein according to the invention orderivative. This is because their discovery enables the inventiveconcept to be put into practice. Such organisms are obtainable bygenerally known techniques, for example by isolating strains from anatural habitat or by screening of gene banks. The nucleotide sequenceshown in SEQ ID NO. 1 may be used, for example, as a screening probe oras an original for the construction of corresponding PCR primers.Analogously, short-chain or complete peptides with amino acid sequencesaccording to SEQ ID NO. 2 may be used to form corresponding antiserawith which corresponding organisms or the proteins released from themcan be identified.

In accordance with the foregoing observations, microorganisms,preferably bacteria, especially gram-positive bacteria including thoseof the genus Bacillus, more particularly Bacillus sp. A 7-7 and mostparticularly Bacillus sp. A 7-7 (DSM 12368), are preferred above all byvirtue of their cultivatability.

Another embodiment of the invention are vectors which contain one of thenucleic acid regions of the second embodiment.

This is because, to use nucleic acids, the DNA is suitably cloned in avector. Such vectors include, for example, those which are derived frombacterial plasmids, from viruses or from bacteriophages or predominantlysynthetic vectors or plasmids with elements of various origins. With theother genetic elements present, vectors are able to establish themselvesas stable units in the respective host cells over several generations.In the context of the invention, it does not matter whether theyestablish themselves extrachromosomally as independent units or areintegrated into a chromosome. Which of the many systems known from theprior art is selected will depend upon the particular individual case.Critical factors in this regard include, for example, the number ofcopies which can be made, the selection systems available, includingabove all resistances to antibiotics, and the cultivatability of thehost cells capable of accommodating the vectors.

The vectors form suitable starting points for molecular-biological andbiochemical investigations of the particular gene or associated proteinand for further developments according to the invention and ultimatelyfor the amplification and production of proteins according to theinvention. They represent embodiments of the present invention insofaras the sequences of the nucleic acid regions according to the inventionpresent lie within the homology range defined in detail in theforegoing.

Preferred embodiments of the present invention are cloning vectors.Besides storage, biological amplification and selection of theinteresting gene, cloning vectors are suitable for the characterizationof the particular gene, for example through the drawing up of arestriction map or sequencing. Cloning vectors are also preferredembodiments of the present invention because they represent atransportable and storable form of the claimed DNA. They are alsopreferred starting points for molecular-biological techniques which arenot restricted to cells, such as the polymerase chain reaction forexample.

Expression vectors have partial sequences which are capable ofreplicating in the host organisms optimized for the production ofproteins and of expressing the gene present in the host organism.Preferred embodiments are expression vectors which themselves carry thegenetic elements necessary for expression. Expression is influenced, forexample, by promoters which regulate the transcription of the gene.Thus, expression can take place through the natural promoter originallylocated before the gene and also after genetically engineered fusionboth through a promoter of the host cell provided on the expressionvector and also through a modified promoter or a totally differentpromoter of another organism.

Preferred embodiments of the invention are expression vectors which canbe regulated through changes in the culture conditions or by addition ofcertain compounds, such as for example the cell density or specialfactors. Expression vectors enable the associated protein to beheterologously produced, i.e. in an organism other than that from whichit can naturally be obtained. Homologous protein production from a hostorganism naturally expressing the gene via a suitable vector also lieswithin the scope of protection of the present invention. This can havethe advantage that natural modification reactions associated with thetranslation can be carried out as well on the protein formed as theywould take place naturally.

Other embodiments of the present invention can be cell-free expressionenzymes where protein biosynthesis is completed in vitro. Expressionsystems such as these are also established in the prior art.

Another embodiment of the present invention are cells which contain oneof the above-defined vectors, more particularly a cloning or expressionvector. This is because, in the course of molecular-biological works asrequired, for example, for mutagenesis, sequencing or storage of thevectors, they are transformed into corresponding cells. Depending on themethod, gram-positive bacteria for example, but especially gram-negativebacteria, can be suitable for this purpose.

Another embodiment are host cells which express a protein or derivativeof the first embodiment or can be stimulated to express that protein orderivative, preferably using an expression vector of the type definedabove.

This is because the preferred in vivo synthesis of an amylolytic enzymeaccording to the invention requires the transfer of the associated geneinto a host cell. Suitable host cells are, in principle, any organisms,i.e. prokaryotes, eukaryotes or cyanophyta. Preferred host cells arethose which are easy to handle genetically, for example as far astransformation with the expression vector and its stable establishmentare concerned, for example single-cell fungi or bacteria. In addition,preferred host cells are distinguished by easy microbiological andbiotechnological handling. This includes, for example, easy cultivation,high growth rates, minimal fermentation media requirements and goodproduction and secretion rates for foreign proteins. The optimalexpression systems for the particular individual case often have to beexperimentally determined from the large number of different systemsavailable in the prior art. In this way, each protein according to theinvention can be obtained from a large number of host organisms.

Preferred embodiments are host cells which can be regulated in theiractivity through genetic regulation elements which are available, forexample, on the expression vector but which can also be present from theoutset in these cells. The host cells in question can be stimulated toexpress, for example by controlled addition of chemical compoundsserving as activators, by changing the cultivation conditions or onreaching a certain cell density. This provides for very economicalproduction of the interesting proteins.

A variant of this experimental principle are expression systems whereadditional genes, for example those made available on other vectors,influence the production of proteins according to the invention. Theymay be modifying gene products or those which are to be purifiedtogether with the protein according to the invention, for example toinfluence its amylolytic function. These may be, for example, otherproteins or enzymes, inhibitors or elements which influence theinteraction with various substrates.

Preferred host cells are prokaryotic or bacterial cells. Bacteria aregenerally distinguished from eucaryotes by shorter generation times andless demanding cultivation conditions. Inexpensive processes for theproduction of proteins according to the invention can thus beestablished.

Host cells and particularly bacteria which secrete the protein orderivative formed into the surrounding medium, so that the expressedproteins according to the invention can be directly purified, areparticularly preferred.

One embodiment of the present invention uses Bacillus sp. A 7-7 (DSM12368) itself for homologously expressing proteins according to theinvention. This can be done, for example, via an introduced vector whichintroduces the already endogenously present gene or modificationsthereof according to the invention into these cells, for example in amultiple number of copies. This can be particularly advantageous if,after its synthesis, the protein is to be subjected to modificationswhich are suitably carried out by the cells in question themselves.

By contrast, heterologous expression is preferred. Gram-positivebacteria, such as actinomycetes or bacilli for example, have no outermembrane so that they release secreted proteins directly into the mediumsurrounding them. Accordingly, bacteria preferred for heterologousexpression include those of the genus Bacillus, more particularly thoseof the species Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus subtilis or Bacillus alcalophilus.

Gram-negative bacteria may also be used for heterologous expression. Intheir case, a large number of proteins are secreted into theperiplasmatic space, i.e. into the compartment between the two membranessurrounding the cells. This can be advantageous for specialapplications. Gram-negative bacteria include, for example, those of thegenus Klebsiella or Escherichia, preferably the species Escherichia coiland more preferably the strains E. coli JM 109, E. coli DH 100B or E.coli DH 12S.

Eukaryotic cells are also suitable for the production of amylolyticproteins according to the invention. Examples include yeasts, such asSaccharomyces or Kluyveromyces. This can be particularly advantageous,for example, when the proteins are to be subjected in connection withtheir synthesis to modifications which such systems allow. Theseinclude, for example, the binding of low molecular weight compounds,such as membrane anchors or oligosaccharides.

All the elements discussed above may be combined into processes forproducing proteins according to the invention. For each proteinaccording to the invention, there are a number of possible combinationsof process steps. They are all practical embodiments of the idea onwhich the present invention is based, namely quantitatively producingrepresentatives of a protein type—defined through the amylolyticfunction and, at the same time, through the high homology to thesequences shown in the sequence protocols—with the aid of the associatedgenetic information. The optimal process has to be experimentallydetermined for each actual individual case.

In principle, the following procedure is adopted: nucleic acidsaccording to the invention, i.e. those which fall within theabove-defined similarity range to the SEQ ID NO. 1 sequence, aresuitably ligated in the form of the DNA in a suitable expression vector.This is transformed into the host cell, for example into cells of aneasy-to-cultivate bacterial strain, which releases the proteins, ofwhich the genes are under the control of corresponding genetic elements,into the surrounding nutrient medium; regulating elements for this canbe made available, for example, by the expression vector. The proteinaccording to the invention can be purified from the surrounding mediumby several purification steps such as, for example, precipitation orchromatography. The expert is able to scale up a system that has beenexperimentally optimized in the laboratory to industrial-scaleproduction.

The most important industrial applications for proteins according to theinvention are listed in the following. Many established industrialapplications for amylolytic enzymes are described in manuals, such asfor example the book by H. Uhlig entitled “Industrial enzymes and theirapplications”, Wiley, New York, 1998. The following list is by no meanscomplete and merely represents a selection of the many theoreticallypossible applications. If it should turn out that individual proteinsaccording to the invention are suitable for additional applications notexpressly claimed herein, those applications are hereby included in thescope of protection of the present invention.

An important application for amylolytic enzymes is their use as activecomponents in detergents/cleaners for cleaning textiles or hardsurfaces. In such applications, the amylolytic activity is used forhydrolytically dissolving carbohydrate-containing, more especiallystarch-like, soils and/or removing them from the substrate. To this end,the enzymes may be used on their own, in suitable media or even indetergents/cleaners. These compositions are distinguished by the factthat the amylolytic enzymes and the other components synergisticallyeffect the elimination of the soils, for example by the hydrolysisproducts of the amylolytic proteins being solubilized by otheringredients of the compositions, such as surfactants for example. Aprotein according to the invention can be used both in compositions forinstitutional or industrial users and in products for the domesticconsumer.

Accordingly, another embodiment of the invention are anydetergents/cleaners which are characterized in that they contain anamylolytic protein according to the invention or derivative thereof.

By this is meant all possible types of cleaning compositions, bothconcentrates and compositions to be used without dilution; for use on acommercial scale, in washing machines or in hand washing or cleaning.Such compositions include, for example, detergents for textiles, carpetsor natural fibers for which the term detergent is used in the presentspecification. They also include, for example detergents for dishwashersor manual dishwashing or cleaners for hard surfaces, such as metals,glass china, ceramic, tiles, stone, painted surfaces, plastics, wood orleather; for these, the term cleaner is used in the presentspecification. Any type of cleaning composition represents an embodimentof the present invention providing it is enriched by a protein accordingto the invention.

Embodiments of the present invention encompass all supply forms of thecompositions according to the invention established in the prior artand/or all appropriate supply forms of the compositions according to theinvention. These include, for example, solid, powder-form, liquid,gel-form or paste-form compositions, optionally consisting of severalphases, compressed or non-compressed. The supply forms also includeextrudates, granules, tablets and pouches packed both in largecontainers and in portions.

Besides an enzyme essential to the invention, the composition accordingto the invention optionally contains other ingredients, such assurfactants, for example nonionic, anionic and/or amphotericsurfactants, and/or bleaching agents and/or builders and optionallyother typical ingredients.

Preferred nonionic surfactants are alkoxylated, advantageouslyethoxylated, more particularly primary alcohols preferably containing 8to 18 carbon atoms and an average of 1 to 12 mmol ethylene oxide (EO)per mol alcohol, in which the alcohol residue may be linear or,preferably, 2-methyl-branched or may contain linear and methyl-branchedresidues in the form of the mixtures typically present in oxoalcoholresidues. However, alcohol ethoxylates containing linear residues ofalcohols of native origin with 12 to 18 carbon atoms, for examplecoconut oil alcohol, palm oil alcohol, tallow alcohol or oleyl alcohol,and an average of 2 to 8 EO per mol alcohol are particularly preferred.Preferred ethoxylated alcohols include, for example, C₁₂₋₁₄ alcoholscontaining 3 EO or 4 EO, C₉₋₁₁ alcohol containing 7 EO, C₁₃₋₁₅ alcoholscontaining 3 EO, 5 EO, 7 EO or 8 EO, C₁₂₋₁₈ alcohols containing 3 EO, 5EO or 7 EO and mixtures thereof, such as mixtures of C₁₂₋₁₄ alcoholcontaining 3 EO and C₁₂₋₁₈ alcohol containing 5 EO. The degrees ofethoxylation mentioned are statistical mean values which, for a specialproduct, may be either a whole number or a broken number. Preferredalcohol ethoxylates have a narrow homolog distribution (narrow rangeethoxylates, NRE). In addition to these nonionic surfactants, fattyalcohols containing more than 12 EO may also be used. Examples of suchfatty alcohols are tallow alcohols containing 14 EO, 25 EO, 30 EO or 40EO.

Another class of preferred nonionic surfactants which are used either assole nonionic surfactant or in combination with other nonionicsurfactants are alkoxylated, preferably ethoxylated or ethoxylated andpropoxylated, fatty acid alkyl esters preferably containing 1 to 4carbon atoms in the alkyl chain, more particularly fatty acid methylesters.

Another class of nonionic surfactants which may be used with advantageare the alkyl polyglycosides (APGs). Suitable alkyl polyglycosidescorrespond to the general formula RO(G)_(z) where R is a linear orbranched, more particularly 2-methyl-branched, saturated or unsaturatedaliphatic radical containing 8 to 22 and preferably 12 to 18 carbonatoms, G is a glycose unit containing 5 or 6 carbon atoms, preferablyglucose. The degree of glycosidation z is between 1.0 and 4.0,preferably between 1.0 and 2.0 and more preferably between 1.1 and 1.4.Linear alkyl polyglucosides, i.e. alkyl polyglycosides in which thepolyglycosyl moiety is a glucose unit and the alkyl moiety is an n-alkylgroup, are preferably used.

Nonionic surfactants of the amine oxide type, for exampleN-cocoalkyl-N,N-dimethylamine oxide and N-tallowalkyl-N,N-dihydroxyethylamine oxide, and the fatty acid alkanolamide type are also suitable. Thequantity in which these nonionic surfactants are used is preferably nomore, in particular no more than half, the quantity of ethoxylated fattyalcohols used.

Other suitable surfactants are polyhydroxyfatty acid amidescorresponding to formula (II):

in which RCO is an aliphatic acyl radical containing 6 to 22 carbonatoms, R¹ is hydrogen, an alkyl or hydroxyalkyl radical containing 1 to4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radicalcontaining 3 to 10 carbon atoms and 3 to 10 hydroxyl groups. Thepolyhydroxyfatty acid amides are known substances which may normally beobtained by reductive amination of a reducing sugar with ammonia, analkylamine or an alkanolamine and subsequent acylation with a fattyacid, a fatty acid alkyl ester or a fatty acid chloride.

The group of polyhydroxyfatty acid amides also includes compoundscorresponding to formula (III):

in which R is a linear or branched alkyl or alkenyl group containing 7to 12 carbon atoms, R¹ is a linear, branched or cyclic alkyl group or anaryl group containing 2 to 8 carbon atoms and R² is a linear, branchedor cyclic alkyl group or an aryl group or an oxyalkyl group containing 1to 8 carbon atoms, C₁₋₄ alkyl or phenyl groups being preferred, and [Z]is a linear polyhydroxyalkyl group, of which the alkyl chain issubstituted by at least two hydroxyl groups, or alkoxylated, preferablyethoxylated or propoxylated, derivatives of that group.

[Z] is preferably obtained by reductive amination of a reduced sugar,for example glucose, fructose, maltose, lactose, galactose, mannose orxylose. The N-alkoxy- or N-aryloxy-substituted compounds may then beconverted into the required polyhydroxyfatty acid amides by, forexample, reaction with fatty acid methyl esters in the presence of analkoxide as catalyst.

Suitable anionic surfactants are, for example, those of the sulfonateand sulfate type. Suitable surfactants of the sulfonate type arepreferably C₉₋₁₃ alkyl benzenesulfonates, olefin sulfonates, i.e.mixtures of alkene and hydroxy-alkane sulfonates, and the disulfonatesobtained, for example, from C₁₂₋₁₈ monoolefins with an internal orterminal double bond by sulfonation with gaseous sulfur trioxide andsubsequent alkaline or acidic hydrolysis of the sulfonation products.Other suitable surfactants of the sulfonate type are the alkanesulfonates obtained from C₁₂₋₁₈ alkanes, for example bysulfochlorination or sulfoxidation and subsequent hydrolysis orneutralization. The esters of α-sulfofatty acids (ester sulfonates), forexample the α-sulfonated methyl esters of hydrogenated coconut oil, palmkernel oil or tallow fatty acids, are also suitable.

Other suitable anionic surfactants are sulfonated fatty acid glycerolesters. Fatty acid glycerol esters in the context of the presentinvention are the monoesters, diesters and triesters and mixturesthereof which are obtained where production is carried out byesterification of a monoglycerol with 1 to 3 mol fatty acid or in thetransesterification of triglycerides with 0.3 to 2 mol glycerol.Preferred sulfonated fatty acid glycerol esters are the sulfonationproducts of saturated fatty acids containing 6 to 22 carbon atoms, forexample caproic acid, caprylic acid, capric acid, myristic acid, lauricacid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)yl sulfates are the alkali metal salts and, inparticular, the sodium salts of the sulfuric acid semiesters of C₁₂₋₁₈fatty alcohols, for example cocofatty alcohol, tallow fatty alcohol,lauryl, myristyl, cetyl or stearyl alcohol, or C₁₀₋₂₀ oxoalcohols andthe corresponding semiesters of secondary alcohols with the same chainlength. Other preferred alk(en)yl sulfates are those with the chainlength mentioned which contain a synthetic, linear alkyl chain based ona petrochemical and which are similar in their degradation behavior tothe corresponding compounds based on oleochemical raw materials. C₁₂₋₁₆alkyl sulfates, C₁₂₋₁₅ alkyl sulfates and C₁₄₋₁₅ alkyl sulfates arepreferred from the point of view of washing technology. Other suitableanionic surfactants are 2,3-alkyl sulfates.

The sulfuric acid monoesters of linear or branched C₇₋₂₁ alcoholsethoxylated with 1 to 6 mol ethylene oxide, such as 2-methyl-branchedC₉₋₁₁ alcohols containing on average 3.5 mol ethylene oxide (EO) orC₁₂₋₁₈ fatty alcohols containing 1 to 4 EO, are also suitable. In viewof their high foaming capacity, they are only used in relatively smallquantities, for example in quantities of 1 to 5% by weight, in cleaners.

Other suitable anionic surfactants are the salts of alkyl sulfosuccinicacid which are also known as sultosuccinates or as sulfosuccinic acidesters and which represent monoesters and/or diesters of sulfosuccinicacid with alcohols, preferably fatty alcohols and, more particularly,ethoxylated fatty alcohols. Preferred sulfosuccinates contain C₈₋₁₈fatty alcohol residues or mixtures thereof. Particularly preferredsulfosuccinates contain a fatty alcohol moiety derived from ethoxylatedfatty alcohols which, considered in isolation, represent nonionicsurfactants (for a description, see below). Of these sulfosuccinates,those of which the fatty alcohol moieties are derived from narrow-rangeethoxylated fatty alcohols are particularly preferred. Alk(en)ylsuccinic acid preferably containing 8 to 18 carbon atoms in thealk(en)yl chain or salts thereof may also be used.

Other suitable anionic surfactants are, in particular, soaps. Suitablesoaps are saturated fatty acid soaps, such as the salts of lauric acid,myristic acid, palmitic acid, stearic acid, hydrogenated erucic acid andbehenic acid, and soap mixtures derived in particular from natural fattyacids, for example coconut oil, palm kernel oil or tallow fatty acids.

The anionic surfactants, including the soaps, may be present in the formof their sodium, potassium or ammonium salts and as soluble salts oforganic bases, such as mono-, di- or triethanolamine. The anionicsurfactants are preferably present in the form of their sodium orpotassium salts and, more preferably, in the form of their sodium salts.

The surfactants may be present in the detergents according to theinvention in a total quantity of preferably 5% by weight to 50% byweight and more particularly 8% by weight to 30% by weight, based on thefinal detergent.

Bleaching agents may be present in accordance with the invention. Amongthe compounds yielding H₂O₂ in water which serve as bleaching agents,sodium percarbonate, sodium perborate tetrahydrate and sodium perboratemonohydrate and are particularly important. Other useful bleachingagents are, for example, peroxopyrophosphates, citrate perhydrates andH₂O₂-yielding peracidic salts or peracids, such as persulfates orpersulfuric acid. The urea peroxohydrate percarbamide, which may bedescribed by the formula H₂N—CO—NH₂.H₂O₂, may also be used. If desired,the compositions may also contain bleaching agents from the group oforganic bleaches, particularly where they are used for cleaning hardsurfaces, for example in dishwashing machines, although in principleorganic bleaches may also be used in laundry detergents. Typical organicbleaching agents are diacyl peroxides, such as dibenzoyl peroxide forexample. Other typical organic bleaching agents are the peroxy acids, ofwhich alkyl peroxy acids and aryl peroxy acids are particularlymentioned as examples. Preferred representatives are peroxybenzoic acidand ring-substituted derivatives thereof, such as alkyl peroxybenzoicacids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate,aliphatic or substituted aliphatic peroxy acids, such as peroxylauricacid, peroxystearic acid, ε-phthalimidoperoxycaproic acid[phthaloiminoperoxyhexanoic acid (PAP)], o-carboxybenzamidoperoxycaproicacid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinates andaliphatic and araliphatic peroxydicarboxylic acids, such as1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacicacid, diperoxybrassylic acid, diperoxyphthalic acids,2-decyldiperoxybutane-1,4-dioic acid,N,N-terephthaloyl-di(6-aminopercaproic acid).

The content of bleaching agents can be from 1 to 40% by weight and, in aparticular embodiment, is from 10 to 20% by weight, perboratemonohydrate or percarbonate advantageously being used. A synergistic useof amylase with percarbonate or amylase with percarboxylic acid isdisclosed in WO 99/63036 and WO 99/63037.

In order to obtain an improved bleaching effect where washing is carriedout at temperatures of 60° C. or lower and particularly in thepretreatment of laundry, the compositions may also contain bleachactivators. Suitable bleach activators are compounds which formaliphatic peroxocarboxylic acids containing preferably 1 to 10 carbonatoms and more preferably 2 to 4 carbon atoms and/or optionallysubstituted perbenzoic acid under perhydrolysis conditions. Substancesbearing O- and/or N-acyl groups with the number of carbon atomsmentioned and/or optionally substituted benzoyl groups are suitable.Preferred bleach activators are polyacylated alkylenediamines, moreparticularly tetraacetyl ethylenediamine (TAED), acylated triazinederivatives, more particularly1,5-diacetyl-2,4-dioxohexa-hydro-1,3,5-triazine (DADHT), acylatedglycolurils, more particularly 1,3,4,6-tetraacetyl glycoluril (TAGU),N-acylimides, more particularly N-nonanoyl succinimide (NOSI), acylatedphenol sulfonates, more particularly n-nonanoyl orisononanoyloxybenzenesulfonate (n- or iso-NOBS), acylatedhydrocarboxylic acids, such as triethyl-O-acetyl citrate (TEOC),carboxylic anhydrides, more particularly phthalic anhydride, isatoicanhydride and/or succinic anhydride, carboxylic acid amides, such asN-methyl diacetamide, glycolide, acylated polyhydric alcohols, moreparticularly triacetin, ethylene glycol diacetate, isopropenyl acetate,2,5-diacetoxy-2,5-dihydrofuran and the enol esters known from Germanpatent applications DE 196 16 693 and DE 196 16 767, acetylated sorbitoland mannitol and the mixtures thereof (SORMAN) described in Europeanpatent application EP 0 525 239, acylated sugar derivatives, moreparticularly pentaacetyl glucose (PAG), pentaacetyl fructose,tetraacetyl xylose and octaacetyl lactose, and acetylated, optionallyN-alkylated glucamine and gluconolactone, triazole or triazolederivatives and/or particulate caprolactams and/or caprolactamderivatives, preferably N-acylated lactams, for example N-benzoylcaprolactam and N-acetyl caprolactam, which are known from Internationalpatent applications WO-A-94/27970, WO-A-94/28102, WO-A-94/28103,WO-A-95/00626, WO-A-95/14759 and WO-A-95/17498. The substitutedhydrophilic acyl acetals known from German patent application DE-A-19616 769 and the acyl lactams described in German patent applicationDE-A-196 16 770 and in International patent application WO-A-95/14075are also preferably used. The combinations of conventional bleachactivators known from German patent application DE-A-44 43 177 may alsobe used. Nitrile derivatives, such as cyanopyridines, nitrile quats, forexample N-alkyl ammonium acetonitriles, and/or cyanamide derivatives mayalso be used. Preferred bleach activators aresodium-4-(octanoyloxy)-benzene sulfonate, n-nonanoyl orisononanoyloxybenzenesulfonate (n- or iso-NOBS),undecenoyloxybenzenesulfonate (UDOBS), sodiumdodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC10) and/or dodecanoyloxybenzenesulfonate (OBS 12) and N-methylmorpholiium acetonitrile (MMA). Bleach activators such as these arepresent in the usual quantities of 0.01 to 20% by weight, preferably inquantities of 0.1% by weight to 15% by weight and more preferably inquantities of 1% by weight to 10% by weight, based on the composition asa whole.

In addition to or instead of the conventional bleach activatorsmentioned above, so-called bleach catalysts may also be incorporated.Bleach catalysts are bleach-boosting transition metal salts ortransition metal complexes such as, for example, manganese-, iron-,cobalt-, ruthenium- or molybdenum-salen complexes or carbonyl complexes.Manganese, iron, cobalt, ruthenium, molybdenum, titanium, vanadium andcopper complexes with nitrogen-containing tripod ligands and cobalt-,iron-, copper- and ruthenium-ammine complexes may also be used as bleachcatalysts, the compounds described in DE 197 09 284 A1 preferably beingused. According to WO 99/63038, acetonitrile derivatives and, accordingto WO 99/63041, bleach-activating transition metal compounds are alsocapable of developing a bleach-activating effect in combination withamylases.

Compositions according to the invention generally contain one or morebuilders, more particularly zeolites, silicates, carbonates, organicco-builders and—providing there are no objections to their use onecological grounds—the phosphates. Phosphates are particularly preferredbuilders in dishwasher detergents.

Suitable crystalline layered sodium silicates correspond to the generalformula NaMSi_(x)O_(2x+1).y H₂O, where M is sodium or hydrogen, x is anumber of 1.6 to 4 and y is a number of 0 to 20, preferred values for xbeing 2, 3 or 4. Crystalline layered silicates such as these aredescribed, for example, in European patent application EP-A-0 164 514.Preferred crystalline layered silicates corresponding to the aboveformula are those in which M is sodium and x assumes the value 2 or 3.Both β- and δ-sodium disilicates Na₂Si₂O₅.y H₂O are particularlypreferred. Such compounds are commercially available, for example, asSKS® (Clariant). Thus SKS-6® is mainly a δ-sodium disilicate with theformula Na₂Si₂O₅.y H₂O while SKS-7® is mainly the β-sodium disilicate.By reaction with acids (for example citric acid or carbonic acid), theδ-sodium disilicate gives kanemite NaHSi₂O₅.H₂O which is marketed asSKS-9® and SKS-10® (Clariant). It can also be of advantage to usechemical modifications of these layered silicates. For example, thealkalinity of the layered silicates can be suitably influenced. Comparedwith the δ-sodium disilicate, phosphate- or carbonate-doped layeredsilicates have modified crystal morphologies, dissolve more quickly andshow increased an calcium binding capacity in relation to δ-sodiumdisilicate. Layered silicates with the general empirical formula xNa₂O.y H₂O.z P₂O₅, in which the ratio of x to y corresponds to a numberof 0.35 to 0.6, the ratio of x to z corresponds to a number of 1.75 to1200 and the ratio of y to z corresponds to a number of 4 to 2,800, aredescribed in patent application DE 19601063. The solubility of thelayered silicates can also be increased by using particularlyfine-particle layered silicates. Compounds of the crystalline layeredsilicates with other ingredients may also be used. Particular mention ismade of compounds with cellulose derivatives, which have advantages inthe disintegrating effect and are used in particular in detergenttablets, and compounds with polycarboxylates, for example citric acid,or polymeric polycarboxylates, for example copolymers of acrylic acid.

Other useful builders are amorphous sodium silicates with a modulus(Na₂O:SiO₂ ratio) of 1:2 to 1:3.3, preferably 1:2 to 1:2.8 and morepreferably 1:2 to 1:2.6 which dissolve with delay and exhibit multiplewash cycle properties. The delay in dissolution in relation toconventional amorphous sodium silicates can have been obtained invarious ways, for example by surface treatment, compounding/compactingor by overdrying. In the context of the invention, the term “amorphous”is also understood to encompass “X-ray amorphous”. In other words, thesilicates do not produce any of the sharp X-ray reflexes typical ofcrystalline substances in X-ray diffraction experiments, but at best oneor more maxima of the scattered X-radiation which have a width ofseveral degrees of the diffraction angle. However, particularly goodbuilder properties may even be achieved where the silicate particlesproduce crooked or even sharp diffraction maxima in electron diffractionexperiments. This may be interpreted to mean that the products havemicrocrystalline regions between 10 and a few hundred nm in size, valuesof up to at most 50 nm and, more particularly, up to at most 20 nm beingpreferred. Compacted amorphous silicates, compounded amorphous silicatesand overdried X-ray-amorphous silicates are particularly preferred.

The finely crystalline, synthetic zeolite containing bound water used inaccordance with the invention is preferably zeolite A and/or zeolite P.Zeolite MAP® (Crosfield) is a particularly preferred P-type zeolite.However, zeolite X and mixtures of A, X and/or P are also suitable.According to the invention, it is preferred to use, for example, acommercially obtainable co-crystallizate of zeolite X and zeolite A (ca.80% by weight zeolite X) which is marketed by CONDEA Augusta S.p.A.under the name of VEGOBOND AX® and which may be described by thefollowing formula:nNa₂O.(1−n)K₂O.Al₂O₃.(2−2.5)SiO₂.(3.5−5.5)H₂O.Suitable zeolites have a moan particle size of less than 10 μm (volumedistribution, as measured by the Coulter Counter Method) and containpreferably 18 to 22% by weight and more preferably 20 to 22% by weightof bound water.

The generally known phosphates may of course also be used as buildersproviding their use should not be avoided on ecological grounds. Amongthe large number of commercially available phosphates, alkali metalphosphates have the greatest importance in the detergent industry,pentasodium triphosphate and pentapotassium triphosphate (sodium andpotassium tripolyphosphate) being particularly preferred.

“Alkali metal phosphates” is the collective term for the alkali metal(more particularly sodium and potassium) salts of the various phosphoricacids, including metaphosphoric acids (HPO₃)_(n) and orthophosphoricacid (H₃PO₄) and representatives of higher molecular weight. Thephosphates combine several advantages: they act as alkalinity sources,prevent lime deposits on machine parts and lime incrustations in fabricsand, in addition, contribute towards the cleaning effect.

Sodium dihydrogen phosphate (NaH₂PO₄) exists as the dihydrate (density1.91 gcm⁻³, melting point 60°) and as the monohydrate (density 2.04gcm⁻³). Both salts are white readily water-soluble powders which, onheating, lose the water of crystallization and, at 200° C., areconverted into the weakly acidic diphosphate (disodium hydrogendiphosphate, Na₂H₂P₂O₇) and, at higher temperatures, into sodiumtrimetaphosphate (Na₃P₃O₉) and Maddrell's salt (see below). NaH₂PO₄shows an acidic reaction. It is formed by adjusting phosphoric acid withsodium hydroxide to a pH value of 4.5 and spraying the resulting “mash”.Potassium dihydrogen phosphate (primary or monobasic potassiumphosphate, potassium biphosphate, KDP), KH₂PO₄, is a white salt with adensity of 2.33 gcm⁻³, has a melting point of 253° [decomposition withformation of potassium polyphosphate (KPO₃)_(x)] and is readily solublein water.

Disodium hydrogen phosphate (secondary sodium phosphate), Na₂HPO₄, is acolorless, readily water-soluble crystalline salt. It exists inwater-free form and with 2 mol (density 2.066 gcm⁻³, water loss at 95°),7 mol (density 1.68 gcm⁻³, melting point 48° with loss of 5H₂O) and 12mol of water (density 1.52 gcm⁻³, melting point 35° with loss of 5H₂O),becomes water-free at 100° and, on fairly intensive heating, isconverted into the diphosphate Na₄P₂O₇. Disodium hydrogen phosphate isprepared by neutralization of phosphoric acid with soda solution usingphenolphthalein as indicator. Dipotassium hydrogen phosphate (secondaryor dibasic potassium phosphate), K₂HPO₄, is an amorphous white saltwhich is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na₃PO₄, consists ofcolorless crystals which have a density of 1.62 gcm⁻³ and a meltingpoint of 73-76° C. (decomposition) as the dodecahydrate, a melting pointof 100° C. as the decahydrate (corresponding to 19-20% P₂O₅) and adensity of 2.536 gcm⁻³ in water-free form (corresponding to 39-40%P₂O₅). Trisodium phosphate is readily soluble in water through analkaline reaction and is prepared by concentrating a solution of exactly1 mole of disodium phosphate and 1 mole of NaOH by evaporation.Tripotassium phosphate (tertiary or tribasic potassium phosphate),K₃PO₄, is a white deliquescent granular powder with a density of 2.56gcm⁻³, has a melting point of 1340° and is readily soluble in waterthrough an alkaline reaction. It is formed, for example, when Thomasslag is heated with coal and potassium sulfate. Despite their higherprice, the more readily soluble and therefore highly effective potassiumphosphates are often preferred to corresponding sodium compounds in thedetergent industry.

Tetrasodium diphosphate (sodium pyrophosphate), Na₄P₂O₇, exists inwater-free form (density 2.534 gcm⁻³, melting point 988°, a figure of880° has also been mentioned) and as the decahydrate (density1.815-1.836 gcm⁻³, melting point 94° with loss of water). Bothsubstances are colorless crystals which dissolve in water through analkaline reaction. Na₄P₂O₇ is formed when disodium phosphate is heatedto >200° or by reacting phosphoric acid with soda in a stoichiometricratio and spray-drying the solution. The decahydrate complexes heavymetal salts and hardness salts and, hence, reduces the hardness ofwater. Potassium diphosphate (potassium pyrophosphate), K₄P₂O₇, existsin the form of the trihydrate and is a colorless hygroscopic powder witha density of 2.33 gcm⁻³ which is soluble iii water, the pH value of a 1%solution at 25° being 10.4.

Relatively high molecular weight sodium and potassium phosphates areformed by condensation of NaH₂PO₄ or KH₂PO₄. They may be divided intocyclic types, namely the sodium and potassium metaphosphates, and chaintypes, the sodium and potassium polyphosphates. The chain types inparticular are known by various different names: fused or calcinedphosphates, Graham's salt, Kurrol's salt and Maddrell's salt. All highersodium and potassium phosphates are known collectively as condensedphosphates.

The industrially important pentasodium triphosphate, Na₅P₃O₁₀ (sodiumtripolyphosphate), is a non-hygroscopic white water-soluble salt whichcrystallizes without water or with 6H₂O and which has the generalformula NaO—[P(O)(ONa)—O]_(n)—Na where n=3. Around 17 g of the salt freefrom water of crystallization dissolve in 100 g of water at roomtemperature, around 20 g at 60° and around 32 g at 100°. After heatingof the solution for 2 hours to 100° around 8% orthophosphate and 15%diphosphate are formed by hydrolysis. In the preparation of pentasodiumtriphosphate, phosphoric acid is reacted with soda solution or sodiumhydroxide in a stoichiometric ratio and the solution is spray-dried.Similarly to Graham's salt and sodium diphosphate, pentasodiumtriphosphate dissolves many insoluble metal compounds (including limesoaps, etc.). Pentapotassium triphosphate, K₅P₃O₁₀ (potassiumtripolyphosphate), is marketed for example in the form of a 50% byweight solution (>23% P₂O₅, 25% K₂O). The potassium polyphosphates arewidely used in the detergent industry. Sodium potassiumtripolyphosphates, which may also be used in accordance with theinvention, also exist. They are formed for example when sodiumtrimetaphosphate is hydrolyzed with KOH:(NaPO₃)₃+2KOH→Na₃K₂P₃O₁₀+H₂O

According to the invention, they may be used in exactly the same way assodium tripolyphosphate, potassium tripolyphosphate or mixtures thereof.Mixtures of sodium tripolyphosphate and sodium potassiumtripolyphosphate or mixtures of potassium tripolyphosphate and sodiumpotassium tripolyphosphate or mixtures of sodium tripolyphosphate andpotassium tripolyphosphate and sodium potassium tripolyphosphate mayalso be used in accordance with the invention.

Organic cobuilders which may be used in the detergents/cleanersaccording to the invention include, in particular, polycarboxylates orpoly-carboxylic acids, polymeric polycarboxylates, polyaspartic acid,polyacetals, optionally oxidized dextrins, other organic cobuilders (seebelow) and phosphonates. These classes of substances are described inthe following.

Useful organic builders are for example, the polycarboxylic acids usablein the form of their sodium salts, polycarboxylic acids in this contextbeing understood to be carboxylic acids which carry more than one acidfunction. These include, for example, citric acid, adipic acid, succinicacid, glutaric acid, malic acid, tartaric acid, maleic acid, fumaricacid, sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA),providing its use is not ecologically unsafe, and mixtures thereof.Preferred salts are the salts of the polycarboxylic acids, such ascitric acid, adipic acid, succinic acid, glutaric acid, tartaric acid,sugar acids and mixtures thereof.

The acids per se may also be used. Besides their building effect, theacids also typically have the property of an acidifying component and,hence, also serve to establish a relatively low and mild pH value indetergents or cleaners unless the pH value obtained by mixing of theother components is required. System-compatible and environmentally safeacids, such as citric acid, acetic acid, tartaric, maleic acid, lacticacid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconicacid and mixtures thereof, are particularly mentioned in this regard.However, mineral acids, particularly sulfuric acid, or bases,particularly ammonium or alkali metal hydroxides, may also be used as pHadjusters. Such adjusters are present in the compositions according tothe invention in quantities of not more than 20% by weight and moreparticularly in quantities of 1.2% by weight to 17% by weight.

Other suitable builders are polymeric polycarboxylates, i.e. for examplethe alkali metal salts of polyacrylic or polymethacrylic acid, forexample those with a relative molecular weight of 500 to 70,000 g/mol.

The molecular weights mentioned in this specification for polymericpolycarboxylates are weight-average molecular weights M_(w) of theparticular acid form which, basically, were determined by gel permeationchromatography (GPC) using a UV detector. The measurement was carriedout against an external polyacrylic acid standard which providesrealistic molecular weight values by virtue of its structural similarityto the polymers investigated. These values differ distinctly from themolecular weights measured against polystyrene sulfonic acids asstandard. The molecular weights measured against polystyrene sulfonicacids are generally higher than the molecular weights mentioned in thisspecification.

Particularly suitable polymers are polyacrylates which preferably have amolecular weight of 2,000 to 20,000 g/mol. By virtue of their superiorsolubility, preferred representatives of this group are the short-chainpolyacrylates which have molecular weights of 2,000 to 10,000 g/mol and,more particularly, 3,000 to 5,000 g/mol.

Also suitable are copolymeric polycarboxylates, particularly those ofacrylic acid with methacrylic acid and those of acrylic acid ormethacrylic acid with maleic acid. Acrylic acid/maleic acid copolymerscontaining 50 to 90% by weight of acrylic acid and 50 to 10% by weightof maleic acid have proved to be particularly suitable. Their relativemolecular weights, based on the free acids, are generally in the rangefrom 2,000 to 70,000 g/mol, preferably in the range from 20,000 to50,000 g/mol and more preferably in the range from 30,000 to 40,000g/mol. The (co)polymeric polycarboxylates may be used either in powderform or in the form of an aqueous solution. The content of (co)polymericpolycarboxylates in the detergents can be from 0.5 to 20% by weight and,more particularly, is from 1 to 10% by weight.

In order to improve solubility in water, the polymers may also containallyl sulfonic acids such as, for example, allyloxybenzene sulfonic acidand methallyl sulfonic acid as monomer.

Other particularly preferred polymers are biodegradable polymers of morethan two different monomer units, for example those which contain saltsof acrylic acid and maleic acid and vinyl alcohol or vinyl alcoholderivatives as monomers or those which contain salts of acrylic acid and2-alkylallyl sulfonic acid and sugar derivatives as monomers.

Other preferred copolymers are those which preferably contain acroleinand acrylic acid/acrylic acid salts or acrolein and vinyl acetate asmonomers.

Other preferred builders are polymeric aminodicarboxylic acids, salts orprecursors thereof. Polyaspartic acids or salts and derivatives thereofwhich have a bleach-stabilizing effect besides their cobuilderproperties are particularly preferred.

Other suitable builders are polyacetals which may be obtained byreaction of dialdehydes with polyol carboxylic acids containing 5 to 7carbon atoms and at least three hydroxyl groups. Preferred polyacetalsare obtained from dialdehydes, such as glyoxal, glutaraldehyde,terephthalaldehyde and mixtures thereof and from polyol carboxylicacids, such as gluconic acid and/or glucoheptonic acid.

Other suitable organic builders are dextrins, for example oligomers orpolymers of carbohydrates which may be obtained by partial hydrolysis ofstarches. The hydrolysis may be carried out by standard methods, forexample acid- or enzyme-catalyzed methods. The end products arepreferably hydrolysis products with average molecular weights of 400 to500,000 g/mol. A polysaccharide with a dextrose equivalent (DE) of 0.5to 40 and, more particularly, 2 to 30 is preferred, the DE being anaccepted measure of the reducing effect of a polysaccharide bycomparison with dextrose which has a DE of 100. Both maltodextrins witha DE of 3 to 20 and dry glucose sirups with a DE of 20 to 37 and alsoso-called yellow dextrins and white dextrins with relatively highmolecular weights of 2,000 to 30,000 g/mol may be used.

The oxidized derivatives of such dextrins are their reaction productswith oxidizing agents which are capable of oxidizing at least onealcohol function of the saccharide ring to the carboxylic acid function.Particularly preferred organic builders for compositions according tothe invention are oxidized starches or derivatives thereof according toEP 472 042, WO 97/25399 and EP 755 944.

Other suitable co-builders are oxydisuccinates and other derivatives ofdisuccinates, preferably ethylenediamine disuccinate.Ethylenediamine-N,N′-disuccinate (EDDS) is preferably used in the formof its sodium or magnesium salts. Glycerol disuccinates and glyceroltrisuccinates are also preferred in this connection. The quantities usedin zeolite-containing and/or silicate-containing formulations are from 3to 15% by weight.

Other useful organic co-builders are, for example, acetylatedhydroxycarboxylic acids and salts thereof which may optionally bepresent in lactone form and which contain at least 4 carbon atoms, atleast one hydroxy group and at most two acid groups.

Another class of substances with co-builder properties are thephosphonates, more particularly hydroxyalkane and aminoalkanephosphonates. Among the hydroxyalkane phosphonates,1-hydroxyethane-1,1-diphosphonate (HEDP) is particularly important as aco-builder. It is preferably used in the form of the sodium salt, thedisodium salt showing a neutral reaction and the tetrasodium salt analkaline reaction (pH 9). Preferred aminoalkane phosphonates areethylenediamine tetramethylene phosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and higher homologs thereof. They arepreferably used in the form of the neutrally reacting sodium salts, forexample as the hexasodium salt of EDTMP or as the hepta- and octasodiumsalts of DTPMP. Of the phosphonates, HEDP is preferably used as abuilder. In addition, the aminoalkane phosphonates have a pronouncedheavy metal binding capacity. Accordingly, it can be of advantage,particularly where the foams also contain bleach, to use aminoalkanephosphonates, more particularly DTPMP, or mixtures of the phosphonatesmentioned.

In addition, any compounds capable of forming complexes with alkalineearth metal ions may be used as co-builders.

The compositions according to the invention may optionally containbuilders in quantities of up to 90% by weight and preferably inquantities of up to 75% by weight. Detergents according to the inventionhave builder contents of in particular 5% by weight to 50% by weight. Inhard surface cleaners according to the invention, particularlydishwasher detergents, the builder content is in particular from 5% byweight to 88% by weight. In a preferred embodiment, such compositionsare free from water-soluble builders. In another preferred embodiment,compositions according to the invention, particularly dishwasherdetergents, contain 20% by weight to 40% by weight water-soluble organicbuilders, more particularly alkali metal citrate, 5% by weight to 15% byweight alkali metal carbonate and 20% by weight to 40% by weight alkalimetal disilicate.

Solvents which may be used in the liquid or gel-form compositions ofdetergents/cleaners belong, for example, to the group of monohydric orpolyhydric alcohols, alkanolamines or glycolethers providing they aremiscible with water in the concentration range indicated. The solventsare preferably selected from ethanol, n- or i-propanol, butanols,ethylene glycol methyl ether, ethylene glycol ethyl ether, ethyleneglycol propyl ether, ethylene glycol moo-n-butyl ether, diethyleneglycol methyl ether, diethylene glycol diethyl ether, propylene glycolmethyl, ethyl or propyl ether, dipropylene glycol monomethyl ormonoethyl ether, diisopropylene glycol monomethyl or monoethyl ether,methoxy, ethoxy or butoxy triglycol, 1-butoxyethoxy-2-propanol,3-methyl-3-methoxybutanol, propylene glycol t-butyl ether and mixturesof these solvents.

Solvents may be present in the liquid or gel-form detergents/cleanersaccording to the invention in quantities of 0.1 to 20% by weight,preferably in quantities below 15% by weight and more particularly inquantities below 10% by weight.

One or more thickeners, for example thickening systems, may be added tothe composition according to the invention in order to adjust itsviscosity. These high molecular weight substances, which are also knownas swelling agents, generally absorb the liquids and swell in theprocess before finally changing into viscous, true or colloidalsolutions.

Suitable thickeners are inorganic or polymeric organic compounds. Theinorganic thickeners include, for example, polysilicic acids, clayminerals, such as montmorillonites, zeolites, silicas and bentonites.The organic thickeners belong to the groups of natural polymers,modified natural polymers and fully synthetic polymers. Examples ofnaturally occurring polymers are agar agar, carrageen, tragacanth, gumarabic, alginates, pectins, polyoses, guar gum, locust bean gum, starch,dextrins, gelatin and casein. Modified natural substances which may beused as thickeners belong, above all, to the group of modified starchesand celluloses and include, for example, carboxymethyl cellulose andother cellulose ethers, hydroxyethyl and hydroxypropyl cellulose and gumethers. Fully synthetic thickeners include polymers, such as polyacrylicand polymethacrylic compounds, vinyl polymers, polycarboxylic acids,polyethers, polyimines, polyamides and polyurethanes.

The thickeners may be used in a quantity of up to 5% by weight,preferably in a quantity of 0.05 to 2% by weight and more particularlyin a quantity of 0.1 to 1.5% by weight, based on the final composition.

The detergents/cleaners according to the invention may optionallycontains sequestering agents, electrolytes and other auxiliaries, suchas optical brighteners, redeposition inhibitors, silver corrosioninhibitors, dye transfer inhibitors, foam inhibitors, abrasives, dyesand/or perfumes, and microbial agents and/or UV absorbers as furtheringredients.

Laundry detergents according to the invention may contain derivatives ofdiaminostilbenedisulfonic acid or alkali metal salts thereof as opticalbrighteners. Suitable optical brighteners are, for example, salts of4,4′-bis-(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)-stilbene-2,2′-disulfonicacid or compounds of similar composition which contain a diethanolaminogroup, a methylamino group, an anilino group or a 2-methoxyethylaminogroup instead of the morpholino group. Brighteners of the substituteddiphenyl styryl type, for example alkali metal salts of4,4′-bis-(2-sulfostyryl)-diphenyl,4,4′-bis-(4-chloro-3-sulfostyryl)-diphenyl or4-(4-chlorostyryl)-4′-(2-sulfostyryl)-diphenyl, may also be present.Mixtures of the brighteners mentioned above may also be used.

The function of redeposition inhibitors is to keep the soil detachedfrom the fibers suspended in the wash liquor. Suitable redepositioninhibitors are water-soluble, generally organic colloids, for examplestarch, glue, gelatin, salts of ether carboxylic acids or ether sulfonicacids of starch or cellulose or salts of acidic sulfuric acid esters ofcellulose or starch. Water-soluble polyamides containing acidic groupsare also suitable for this purpose. Starch derivatives other than thosementioned above, for example aldehyde starches, etc., may also be used.Cellulose ethers, such as carboxymethyl cellulose (sodium salt), methylcellulose, hydroxyalkyl cellulose, and mixed ethers, such as methylhydroxyethyl cellulose, methyl hydroxypropyl cellulose, methylcarboxymethyl cellulose and mixtures thereof, are preferably used, forexample in quantities of 0.1 to 5% by weight, based on the composition.

In order to protect silverware against corrosion, silver corrosioninhibitors may be used in dishwashing detergents according to theinvention. Silver corrosion inhibitors are known from the prior art andinclude, for example, benzotriazoles, iron (III) chloride and CoSO₄. Asknown from European patent EP 0 736 084 B1, for example, silvercorrosion inhibitors particularly suitable for use together with enzymesare manganese, titanium, zirconium, hafnium, vanadium, cobalt or ceriumsalts and/or complexes in which the metals mentioned are present in oneof the oxidation numbers II, II, IV, V or VI. Examples of such compoundsare MnSO₄, V₂O₅, V₂O₄, VO₂, TIOSO₄, K₂TiF₆, K₂ZrF₆, Co(NO₃)₂, Co(NO₃)₃and mixtures thereof.

Soil release agents or soil repellents are generally polymers which,when used in a laundry detergent, provide the laundry fibers withsoil-repelling properties and/or support the soil suspending capacity ofthe other ingredients of the detergent. A comparable effect can also beobserved where they are used in hard surface cleaners.

Particularly effective and long-established soil release agents arecopolyesters containing dicarboxylic acid, alkylene glycol andpolyalylene glycol units. Examples are copolymers or copolymers ofpolyethylene terephthalate and polyoxyethylene glycol (DT 16 17 141 orDT 22 00 911). DE-OS 2253063 mentions acidic compositions containinginter alia a copolymer of a dibasic carboxylic acid and an alkylene orcycloalkylene polyglycol. Polymers of ethylene terephthalate andpolyethylene oxide terephthalate and their use in detergents isdescribed in DE-PS 28 57 292 and DE-PS 33 24 258 and in EP 0 253 567.European patent EP 066 944 relates to compositions containing acopolyesters of ethylene glycol, polyethylene glycol, aromaticdicarboxylic acid and sulfonated aromatic dicarboxylic acid in certainmolar ratios. European patent EP 0 185 427 describes methyl- orethyl-terminated polyesters containing ethylene and/or propyleneterephthalate units and polyethylene oxide terephthalate units anddetergents which contain such a soil-release polymer. European patentrelates to a polyester which, besides oxyethylene groups andterephthalic acid units, also contains substituted ethylene units andglycerol units. European patent EP 0 241 985 describes polyesters which,besides oxyethylene groups and terephthalic acid units, contain1,2-propylene, 1,2-butylene and/or 3-methoxy-1,2-propylene groups andglycerol units and which are terminated by C₁₋₄ alkyl groups. Europeanpatent application EP 0 272 033 describes at least partly C₁₋₄ alkyl- oracyl-terminated polyesters containing polypropylene terephthalate andpolyoxyethylene terephthalate units. European patent EP 0 274 907describes sulfoethyl-terminated terephthalate-containing soil-releasepolyesters. According to European patent application EP 0 357 280,soil-release polyesters containing terephthalate, alkylene glycol andpoly-C₂₋₄-glycol units are produced by sulfonation of unsaturatedterminal groups. International patent application WO 95/32232 relates toacidic aromatic soil-release polyesters. International patentapplication WO 97/31085 describes soil repellents for cotton fabricswhich contain several functional units: a first unit, which may becationic for example, is capable of adsorption onto the cotton surfaceby electrostatic interaction, and a second unit which is hydrophobic isresponsible for the active substance remaining at the water/cottoninterface.

Dye transfer inhibitors suitable for use in laundry detergents accordingto the invention include, in particular, polyvinyl pyrrolidones,polyvinyl imidazoles, polymeric N-oxides, such aspoly-(vinylpyridine-N-oxide) and copolymers of vinyl pyrrolidone withvinyl imidazole.

Where the compositions are used in machine cleaning processes, it can beof advantage to add typical foam inhibitors to them. Suitable foaminhibitors are, for example, soaps of natural or synthetic origin whichhave a high percentage content of C₁₈₋₂₄ fatty acids. Suitablenon-surface-active foam inhibitors are, for example, organopolysiloxanesand mixtures thereof with microfine, optionally silanized, silica andalso paraffins, waxes, microcrystalline waxes and mixtures thereof withsilanized silica or bis-stearyl ethylenediamide. Mixtures of differentfoam inhibitors, for example mixtures of silicones, paraffins and waxes,may also be used with advantage. The foam inhibitors, more particularlysilicone- and/or paraffin-containing foam inhibitors, are preferablyfixed to a granular water-soluble or water-dispersible support. Mixturesof paraffins and bis-stearyl ethylenediamides are particularlypreferred.

In addition, a hard surface cleaner according to the invention maycontain abrasive constituents, more particularly from the groupconsisting of silica flours, wood flours, plastic powders, chalks andglass microbeads and mixtures thereof. Abrasives are present in thecleaners according to the invention is quantity of preferably not morethan 20% by weight and more particularly in quantities of 5% to 15% byweight.

Dyes and perfumes are added to the detergents/cleaners according to theinvention to improve the aesthetic impression created by the productsand to provide the consumer not only with the required washing andcleaning performance but also with a visually and sensorially “typicaland unmistakable” product. Suitable perfume oils or fragrances includeindividual perfume compounds, for example synthetic products of theester, ether, aldehyde, ketone, alcohol and hydrocarbon type. Perfumecompounds of the ester type are, for example, benzyl acetate,phenoxyethyl isobutyrate, p-tert.butyl cyclohexyl acetate, linalylacetate, dimethyl benzyl carbinyl acetate, phenyl ethyl acetate, linalylbenzoate, benzyl formate, ethyl methyl phenyl glycinate, allylcyclohexyl propionate, styrallyl propionate and benzyl salicylate. Theethers include, for example, benzyl ethyl ether; the aldehydes include,for example, the linear alkanals containing 8 to 18 carbon atoms,citral, citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde,hydroxycitronellal, lilial and bourgeonal; the ketones include, forexample, the ionones, α-isomethyl ionone and methyl cedryl ketone; thealcohols include anethol, citronellol, eugenol, geraniol, linalool,phenyl ethyl alcohol and terpineol and the hydrocarbons include, aboveall, the terpenes, such as limonene and pinene. However, mixtures ofvarious perfumes which together produce an attractive perfume note arepreferably used. Perfume oils such as these may also contain naturalperfume mixtures obtainable from vegetable sources, for example pine,citrus, jasmine, patchouli, ruse or ylang-ylang oil. Also suitable areclary oil, chamomile oil, clove oil, melissa oil, mint oil, cinnamonleaf oil, lime blossom oil, juniper berry oil, vetiver oil, olibanumoil, galbanum oil and ladanum oil and orange blossom oil, neroli oil,orange peel oil and sandalwood oil. The dye content ofdetergents/cleaners is usually below 0.01% by weight while perfumes canmake up as much as 2% by weight of the formulation as a whole.

The perfumes may be directly incorporated in the detergents/cleaners,although it can also be of advantage to apply the fragrances to supportswhich strengthen the adherence of the perfume to the articles beingwashed/cleaned and which provide treated textiles in particular with along-lasting fragrance through a slower release of the perfume. Suitablesupport materials are, for example, cyclodextrins, thecyclodextrin/perfume complexes optionally being coated with otherauxiliaries. Another preferred support for perfumes is the describedzeolite X which is also capable of absorbing perfumes instead of or inadmixture with surfactants. Accordingly, detergents/cleaners containingthe described zeolite X and perfumes preferably absorbed at least partlyon the zeolite are preferred.

Preferred dyes which the expert will find no difficulty in selectinghave high stability in storage, are unaffected by the other ingredientsof the composition and by light and do not show pronounced substantivitytowards textile fibers so as not to color them.

To control microorganisms, detergents/cleaners may contain antimicrobialagents. Depending on the antimicrobial spectrum and the actionmechanism, antimicrobial agents are classified as bacteriostatic agentsand bactericides, fungistatic agents and fungicides, etc. Importantrepresentatives of these groups are, for example, benzalkoniumchlorides, alkylaryl sulfonates, halophenols and phenol mercuriacetate.In the context of the teaching according to the invention, theexpressions “antimicrobial activity” and “antimicrobial agent” have theusual meanings as defined, for example, by K. H. Wallhäuβer in “Praxisder Sterilisation, Desinfektion-Konservierung:Keimidentifizierung-Betriebshygiene” (5th Edition, Stuttgart/New York:Thieme, 1995), any of the substances with antimicrobial activitydescribed therein being usable. Suitable antimicrobial agents arepreferably selected from the groups of alcohols, amines, aldehydes,antimicrobial acids and salts thereof, carboxylic acid esters, acidamides, phenols, phenol derivatives, diphenyls, diphenylalkanes, ureaderivatives, oxygen and nitrogen acetals and formals, benzamidines,isothiazolines, phthalimide derivatives, pyridine derivatives,antimicrobial surface-active compounds, guanidines, antimicrobialamphoteric compounds, quinolines, 1,2-dibromo-2,4-dicyanobutane,iodo-2-propyl butyl carbamate, iodine, iodophores, peroxo compounds,halogen compounds and mixtures of the above.

The antimicrobial agent may be selected from ethanol, n-propanol,i-propanol, butane-1,3-diol, phenoxyethanol, 1,2-propylene glycol,glycerol, undecylenic acid, benzoic acid, salicylic acid, dihydraceticacid, o-phenylphenol, N-methyl morpholine acetonitrile (MMA),2-benzyl-4-chlorophenol, 2,2′-dimethylene-bis-(6-bromo-4-chlorophenol),4,4′-dichloro-2′-hydroxydiphenyl ether (Dichlosan),2,4,4′-trichloro-2′-hydroxydiphenyl ether (Trichlosan), chlorohexidine,N-(4-chlorophenyl)-N-3,4-dichlorophenyl)-urea,N,N′-(1,10-decanediyldi-1-pyridinyl-4-ylidene)-bis-(1-octanamine)-dihydrochloride,N,N′-bis-(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimidoamide, glucoprotamines, antimicrobial surface-active quaternarycompounds, guanidines, including the bi- and polyguanidines such as, forexample, 1,6-bis-(2-ethylhexylbiguanidohexane)-dihydrochloride,1,6-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)-hexane tetrahydrochloride,1,6-di-(N₁, N₁′-phenyl-N₁,N₁-methyldiguanido-N₅, N₅′)-hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)-hexanedihydrochloride,1,6-di-(N₁,N₁′-2,6-dichlorophenyldiguanido-N₅,N₅′)-hexanedihydrochloride,1,6-di-[N₁N₁′-β-(p-methoxyphenyl)-diguanido-N₅,N₅′]-hexanedihydrochloride, 1,6-di-(N₁N₁′-α-methyl-β-phenyldiguanido-N₅,N₅′)-hexanedihydrochloride, 1,6-di-(N₁,N₁′-p-nitrophenyldiguanido-N₅,N₅′)-hexanedihydrochloride, ω:ω-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)-di-n-propylether dihydrochloride,ω:ω′-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-di-n-propyl ethertetrahydrochloride,1,6-di-(N₁,N₁′-2,4-dichlorophenyldiguanido-N₅,N₅′)-hexanetetrahydrochloride,1,6-di-(N₁,N₁′-p-methylphenyldiguanido-N₅,N₅′)-hexanedihydrochloride,1,6-di-(N₁,N₁′-2,4,5-trichlorophenyldiguanido-N₅,N₅′)-hexanetetrahydrochloride,1,6-di-[N₁,N₁′-α-(p-chlorophenyl)-ethyldiguanido-N₅,N₅′]-hexanedihydrochloride, ω:ω-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-m-xylenedihydrochloride,1,12-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-dodecanedihydrdochloride, 1,10-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)-decanetetrahydrochloride, 1,12-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)-dodecanetetrahydrochloride,1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)-hexane dihydrochloride,1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)-hexanetetrahydrochloride, ethylene-bis-(1-tolylbiguanide),ethylene-bis-(p-tolylbiguanide),ethylene-bis-(3,5-dimethylphenylbiguanide),ethylene-bis-(p-tert.amylphenylbiguanide),ethylene-bis-(nonylphenylbiguanide), ethylene-bis-(phenylbiguanide),ethylene-bis-(N-butylphenylbiguanide),ethylene-bis-(2,5-diethoxyphenylbiguanide),ethylene-bis-(2,4-dimethylphenylbiguanide),ethylene-bis-(o-diphenylbiguanide),ethylene-bis-(mixed-amylnaphthylbiguanide),N-butylethylene-bis-(phenylbiguanide),trimethylene-bis-(o-tolylbiguanide),N-butyltrimethylene-bis-(phenylbiguanide) and the corresponding salts,such as acetates, gluconates, hydrochlorides, hydrobromides, citrates,bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates,phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates,salicylates, maleates, tartrates, fumarates, ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates, arginates,pyromellitates, tetracarboxybutyrates, benzoates, glutarates,monofluorophosphates, perfluoropropionates and mixtures thereof.Halogenated xylene and cresol derivatives, such as p-chloro-m-cresol orp-chloro-m-xylene, and natural antimicrobial agents of vegetable origin(for example from spices or herbs), animal and microbial origin are alsosuitable. Preferred antimicrobial agents are antimicrobialsurface-active quaternary compounds, a natural antimicrobial agent ofvegetable origin and/or a natural antimicrobial agent of animal originand, most preferably, at least one natural antimicrobial agent ofvegetable origin from the group comprising caffeine, theobromine andtheophylline and essential oils, such as eugenol, thymol and geraniol,and/or at least one natural antimicrobial agent of animal origin fromthe group comprising enzymes, such as protein from milk, lysozyme andlactoperoxidase and/or at least one antimicrobial surface-activequaternary compound containing an ammonium, sulfonium, phosphonium,iodonium or arsonium group, peroxo compounds and chlorine compounds.Substances of microbial origin, so-called bacteriozines, may also beused.

The quaternary ammonium compounds (QUATS) suitable as antimicrobialagents have the general formula (R¹)(R²)(R³)(R⁴)N⁺X⁻, in which R¹ to R⁴may be the same or different and represent C₁₋₂₂ alkyl groups, C₇₋₂₈aralkyl groups or heterocyclic groups, two or—in the case of an aromaticcompound, such as pyridine—even three groups together with the nitrogenatom forming the heterocycle, for example a pyridinium or imidazoliniumcompound, and X⁻ represents halide ions, sulfate ions, hydroxide ions orsimilar anions. In the interests of optimal antimicrobial activity, atleast one of the substituents preferably has a chain length of 8 to 18and, more preferably, 12 to 16 carbon atoms.

QUATS can be obtained by reaction of tertiary amines with alkylatingagents such as, for example, methyl chloride, benzyl chloride, dimethylsulfate, dodecyl bromide and also ethylene oxide. The alkylation oftertiary amines with one long alkyl chain and two methyl groups isparticularly simple. The quaternization of tertiary amines containingtwo long chains and one methyl group can also be carried out under mildconditions using methyl chloride. Amines containing three long alkylchains or hydroxy-substituted alkyl chains lack reactivity and arepreferably quaternized with dimethyl sulfate.

Suitable QUATS are, for example, benzalkonium chloride(N-alkyl-N,N-dimethylbenzyl ammonium chloride, CAS No. 8001-54-5),benzalkon B (m,p-dichlorobenzyl dimethyl-C₁₂-alkyl ammonium chloride,CAS No. 58390-78-6), benzoxonium chloride(benzyldodecyl-bis-(2-hydroxyethyl)-ammonium chloride), cetrimoniumbromide (N-hexadecyl-N,N-trimethyl ammonium bromide, CAS No. 57-09-0),benzetonium chloride(N,N-dimethyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy]-ethyl]-benzylammonium chloride, CAS No. 121-54-0), dialkyl dimethyl ammoniumchlorides, such as di-n-decyldimethyl ammonium chloride (CAS No.7173-51-5-5), didecyldimethyl ammonium bromide (CAS No. 2390-68-3),dioctyl dimethyl ammonium chloride, 1-cetylpyridinium chloride (CAS No.123-03-5) and thiazoline iodide (CAS No. 15764-48-1) and mixturesthereof. Particularly preferred QUATS are the benzalkonium chloridescontaining C₈₋₁₈ alkyl groups, more particularly C₁₂₋₁₄ alkyl benzyldimethyl ammonium chloride.

Benzalkonium halides and/or substituted benzalkonium halides arecommercially obtainable, for example, as Barquat® from Lonza, Marquat®from Mason, Variquat® from Witco/Sherex and Hyamine® from Lonza and asBardac® from Lonza. Other commercially obtainable antimicrobial agentsare N-(3-chloroallyl)-hexaminium chloride, such as Dowicide® andDowicil® from Dow, benzethonium chloride, such as Hyamine® 1622 fromRohm & Haas, methyl benzethonium chloride, such as Hyamine® 10X fromRohm & Haas, cetyl pyridinium chloride, such as cepacolchloride fromMerrell Labs.

The antimicrobial agents are used in quantities of 0.0001% by weight to1% by weight, preferably in quantities of 0.001% by weight to 0.8% byweight, more preferably in quantities of 0.005 to 0.3% by weight andmost preferably in quantities of 0.01 to 0.2% by weight.

In addition, the compositions may optionally contain UV absorbers whichare absorbed onto the treated textiles and improve the light stabilityof the fibers and/or the light stability of the other formulationingredients. UV absorbers are organic substances (light filters) whichare capable of absorbing ultraviolet rays and of releasing the energyabsorbed in the form of longer-wave radiation, for example heat.

Compounds which possess these desired properties are, for example, thecompounds which act by radiationless deactivation and derivatives ofbenzophenone with substituents in the 2- and/or 4-position. Othersuitable UV absorbers are substituted benzotriazoles,3-phenyl-substituted acrylates (cinnamic acid derivatives, optionallywith cyano groups in the 2-position), salicylates, organic Ni complexesand natural substances, such as umbelliferone and the body's ownurocanic acid. Particular significance attaches to the biphenyl and,above all, stilbene derivatives described, for example, EP 0728740 Awhich are commercially available as Tinosorb® FD and Tinosorb® FR exCiba. Suitable UV-B absorbers include 3-benzylidene camphor or3-benzylidene norcamphor and derivatives thereof, for example3-(4-methylbenzylidene)-camphor as described in EP-B1 0693471;4-aminobenzoic acid derivatives, preferably 4-(dimethylamino)-benzoicacid-2-ethylhexyl ester, 4-(dimethylamino)-benzoic acid-2-octyl esterand 4-(dimethylamino)-benzoic acid amyl ester; esters of cinnamic acid,preferably 4-methoxycinnamic acid-2-ethylhexyl ester, 4-methoxycinnamicacid propyl ester, 4-methoxycinnamic acid isoamyl ester,2-cyano-3,3-phenylcinnamic acid-2-ethylhexyl ester (Octocrylene); estersof salicylic acid, preferably salicylic acid-2-ethylhexyl ester,salicylic acid-4-isopropylbenzyl ester, salicylic acid homomethyl ester;derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid,preferably 4-methoxybenzmalonic acid di-2-ethylhexyl ester; triazinederivatives such as, for example,2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine and OctylTriazone as described in EP 0818450 A1 or Dioctyl Butamido Triazone(Uvasorb® HEB): propane-1,3-diones such as, for example,1-(4-tert.butylphenyl)-3-(4′-methoxyphenyl)-propane-1,3-dione;ketotricyclo(5.2.1.0)decane derivatives as described in EP 0694521 B1.Other suitable UV-B absorbers are 2-phenylbenzimidazole-5-sulfonic acidand alkali metal, alkaline earth metal, ammonium, alkylammonium,alkanolammonium and glucammonium salts thereof; sulfonic acidderivatives of benzophenones, preferably2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and salts thereof;sulfonic acid derivatives of 3-benzylidene camphor such as, for example,4-(2-oxo-3-bornylidenemethyl)-benzene sulfonic acid and2-methyl-5-(2-oxo-3-bornylidene)-sulfonic acid and salts thereof.

Typical UV-A filters are, in particular, derivatives of benzoyl methanesuch as, for example,1-(4′-tert.butylphenyl)-3-(4′-methoxyphenyl)-propane-1,3-dione,4-tert.butyl-4′-methoxydibenzoyl methane (Parsol 1789),1-phenyl-3-(4′-isopropylphenyl)-propane-1,3-dione and the enaminecompounds described in DE 19712033 A1 (BASF). The UV-A and UV-B filtersmay of course also be used in the form of mixtures. Besides the solublesubstances mentioned, insoluble light-blocking pigments, i.e. finelydispersed, preferably “nanoized” metal oxides or salts, may also be usedfor this purpose. Examples of suitable metal oxides are, in particular,zinc oxide and titanium dioxide and also oxides of iron, zirconiumoxide, silicon, manganese, aluminium and cerium and mixtures thereof.Silicates (talcum), barium sulfate and zinc stearate may be used assalts. The oxides and salts are used in the form of the pigments forskin-care and skin-protecting emulsions and decorative cosmetics. Theparticles should have a mean diameter of less than 100 nm, preferablybetween 5 and 50 nm and more preferably between 15 and 30 nm. They maybe spherical in shape although ellipsoidal particles or othernon-spherical particles may also be used. The pigments may also besurface-treated, i.e. hydrophilicized or hydrophobicized. Typicalexamples are coated titanium dioxides, for example Titandioxid T 805(Degussa) and Eusolex® T2000 (Merck). Suitable hydrophobic coatingmaterials are, above all, silicones and, among these, especiallytrialkoxyoctylsilanes or simethicones. Micronized zinc oxide ispreferably used. Other suitable UV filters can be found in P. Finkel'sreview in SÖFW-Journal 122, 543 (1996).

The UV absorbers are normally used in quantities of 0.01% by weight to5% by weight and preferably 0.03% by weight to 1% by weight.

Proteins according to the invention and/or other proteins may alsorequire special protection in detergents and cleaners. Compositionsaccording to the invention may contain stabilizers for this purpose.

One group of stabilizers is reversible protease inhibitors whichdissociate off on dilution of the composition in the wash liquor.Benzamidine hydrochloride and leupeptin are established for thispurpose. Borax, boric acids, boron acids or salts or esters thereof,including above all phenylboron acids orthosubstituted by aromaticgroups, for example in accordance with WO 95/12655, metasubstituted byaromatic groups in accordance with WO 92/19707 and parasubstituted byaromatic groups in accordance with U.S. Pat. No. 5,972,873 or salts oresters thereof, are often used for this purpose. WO 98/13460 and EP 583534 disclose peptide aldehydes, i.e. oligopeptides with a reducedC-terminus, especially those of 2-50 monomers, for the reversibleinhibition of detergent proteases. The peptidic reversible proteaseinhibitors include inter alia ovomucoid (WO 93/00418). WO 00/01826, forexample, discloses specific reversible peptide inhibitors for theprotease subtilisin for use in protease-containing compositions while WO00/01831 discloses corresponding fusion proteins of protease andinhibitor.

Other enzyme stabilizers are aminoalcohols, such as mono-, di-,triethanol- and -propanolamine and mixtures thereof, aliphaticcarboxylic acids up to C₁₂ as known, for example, from EP 0 378 261 andWO 97/05227, such as succinic acid, other dicarboxylic acids or salts ofthe acids mentioned. End-capped fatty acid amide alkoxylates aredisclosed for this purpose in German patent application DE 19650537.Certain organic acids used as builders are additionally capable ofstabilizing an enzyme present, as disclosed in WO 97/18287.

Lower aliphatic alcohols, but above all polyols such as, for example,glycerol, ethylene glycol, propylene glycol or sorbitol are otherfrequently used enzyme stabilizers. According to a more recentapplication (EP 0 965 268), diglycerophosphate also stabilizes againstdenaturing by physical influences. Calcium salts, for example calciumacetate or the calcium formate disclosed for this purpose in EP 0 028865, and magnesium salts, for example according to European patentapplication EP 0 378 262, are also used.

Polyamide oligomers (WO 99/43780) or polymeric compounds, such as lignin(WO 97/00932), water-soluble vinyl copolymers (EP 828 762) or, asdisclosed in EP 702 712, cellulose ethers, acrylic polymers and/orpolyamides stabilize the enzyme preparation inter alia against physicalinfluences or pH variations. Polymers containing polyamine-N-oxide (EP587 550 and EP 581 751) simultaneously act as enzyme stabilizers and asdye transfer inhibitors. Other polymer stabilizers are the linear C₈₋₁₈polyoxyalkylene disclosed besides other constituents in WO 97/05227.Alkyl polyglycosides could stabilize the enzymatic components of thecomposition according to the invention and even enhance theirperformance, as disclosed in WO 97/43377 and WO 98/45396. CrosslinkedN-containing compounds, as disclosed in WO 98/17764, perform a dual roleas soil release agents and as enzyme stabilizers. Hydrophobic nonionicpolymer in admixture with other stabilizers according to WO 97/32958 hasa stabilizing effect on a cellulase so that these or similarconstituents could also be suitable for the enzyme essential to theinvention.

Reducing agents and antioxidants, as disclosed inter alia in EP 780 466,increase the stability of the enzymes to oxidative disintegration.Sulfur-containing reducing agents are known, for example, from EP 0 080748 and EP 0 080 223. Other examples are sodium sulfite (EP 533 239) andreducing sugars (EP 656 058).

In many cases, combinations of stabilizers are also used, for examplethe combination of polyols, boric acid and/or borax in WO 96/31589, thecombination of boric acid or borate, reducing salts and succinic acid orother dicarboxylic acids in EP 126 505 or the combination of boric acidor borate with polyols or polyamino compounds and with reducing salts asdisclosed in EP 080 223. According to WO 98/13462, the effect ofpeptide/aldehyde stabilizers is enhanced by combination with boric acidand/or boric acid derivatives and polyols and, according to WO 98/13459is further enhanced by the additional use of calcium ions.

Compositions with stabilized enzyme activities represent preferredembodiments of the present invention. Compositions containing enzymesstabilized in several of the ways mentioned are particularly preferred.

Compositions according to the invention contain proteins or derivativesessential to the invention in quantities of preferably 0.000001% byweight to 5% by weight and, with increasing preference, in quantities of0.00005 to 4% by weight, 0.00001 to 3% by weight, 0.0001 to 2% by weightand, in a most particularly preferred embodiment, in quantities of 0.001to 1% by weight. The protein concentration can be determined by knownmethods, for example the BCA method (bicinchonic acid;2,2′-biquinolyl-4,4′-dicarboxylic acid) or the biuret method (A. G.Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem. 177, (1948),pp. 751-766).

Besides the protein essential to the invention, compositions accordingto the invention may contain other amylolytic enzymes, more particularlyα-amylases. These may also include the enzymes established for use indetergents/cleaners which were mentioned at the beginning. Examples ofcommercially available amylases are BAN®, Termamyl®, Purastar®,Amylase-LT®, Maxamyl®, Duramyl® and/or Purafect® Ox-Am. This isadvisable when the various enzymes are capable of complementing oneanother. Such complementing may occur, for example, in a regulatorysense, for example through mutual activation or inactivation. It mayarise, for example, through at least a part of the enzyme essential tothe invention, which is not homologous to the known α-amylases, havingan influence on the amylolytic activities not essential to theinvention.

However, the joint use can also be appropriate in view of differingsubstrate specificities. Both are embodiments of the present invention.

It can be of advantage, particularly with chemically diverse soils, touse amylolytic enzymes in detergents/cleaners together with otherdetersive enzymes and/or enzymes with cleaning activity. Accordingly,detergents/cleaners characterized by other enzymes besides a proteinaccording to the invention represent preferred embodiments of thepresent invention.

Besides other amylases, for example proteases, these other enzymesinclude, for example, lipases, cutinases, esterases, pullulanases,cellulases, hemicellulases and/or xylanases and mixtures thereof.Proteases, lipases, β-glucanases and/or cellulases are particularlypreferred. Other enzymes add to the cleaning performance ofcorresponding compositions through their particular specific enzymaticactivity. These include, for example, oxidoreductases or peroxidases ascomponents of enzymatic bleaching systems, laccases (WO 00/39306),β-glucanases (WO 99/06515 and WO 99/06516) or pectin-dissolving enzymes(WO 00/42145) which are used in particular in specialty detergents.

Examples of commercially obtainable enzymes for use in compositionsaccording to the invention are proteases, such as subtilisin BPN′,Properase®, BLAP®, Optimase®, Opticlean®, Maxatase®, Maxacal®, Maxapem®,Alcalase®, Esperase®, Savinase®, Durazym®, Everlase® and/or Purafect®Gor Purafect®OxP, and lipases, such as Lipolase®, Lipomax®, Lumafast®and/or Lipozym®.

The protease activity in such compositions may be determined by themethod described in Tenside, Vol. 7 (1970), pp. 125-132. It is expressedaccordingly, in PU (protease units). The protease activity of preferredcompositions can be as high as 1,500,000 protease units per grampreparation (PU, as determined by the method described in Tenside, Vol.7 (1970), pp. 125-132).

So far as their isolation is concerned, suitable enzymes are, above all,those obtained from microorganisms, such as bacteria or fungi, forexample from Bacillus subtilis, Bacillus licheniformis, Streptomycesgriseus, Humicola lanuginosa, Humicola insolens, Pseudomonaspseudoalcaligenes or Pseudomonas cepacia, more particularly the enzymemixtures formed naturally by these strains or mixtures with otherstrains. They are isolated in known manner by fermentation processesfrom suitable microorganisms which are described, for example, in GermanOffenlegungsschrifts DE 1940488 and DE 2121397, in US patents U.S. Pat.No. 3,623,957 and U.S. Pat. No. 4,264,738, in European patentapplication EP 006 638 and in International patent application WO91/02792.

These optional additional enzymes may also be adsorbed onto carriersand/or encapsulated in membrane materials to protect them againstpremature inactivation, as described for example in European patent EP 0564 476 or in International patent application WO 94/23006. They arepresent in detergents in quantities of preferably up to 10% by weightand more particularly from 0.2% by weight to 2% by weight, enzymesstabilized against oxidative degradation—as known, for example, fromInternational patent application WO 94/18314—being particularlypreferred.

Compositions according to the invention may consist of several phases,for example in order to release the active ingredients presentseparately from one another in time or space. The phases may be invarious aggregate states but, in a particular embodiment, are two phasesin the same aggregate state.

Compositions according to the invention which are made up of severalsolid components may readily be produced by mixing various powders orgranules having various ingredients and/or different release behaviorwith one another in an overall loose form. The production of solid,single-phase or multi-phase compositions according to the invention canbe carried out in known manner, for example by spray drying orgranulation, the enzymes and optionally other heat-sensitiveingredients, such as bleaching agents for example, optionally beingseparately added at a later stage. Compositions according to theinvention with a high bulk density, more particularly in the range from650 to 950 g/l, are preferably produced by a process involving anextrusion step known from EP 0 486 592. Another preferred productionprocess based on granulation is described in European patent EP 0 642576.

Proteins can be used, for example, in dried, granulated, or encapsulatedform or in encapsulated and additionally dried form for soliddetergents/cleaners. They may be added separately, i.e. as a separatephase, or together with other constituents in the same phase without orwithout compacting. If microencapsulated enzymes are to be processed insolid form, the water can be removed from the aqueous solutionsresulting from working up, for example by spray drying, centrifuging orresolubilization. The particles obtained in this way normally have aparticle size of 50 to 200 μm.

The encapsulated form is appropriate for protecting the enzymes fromother constituents, such as bleaching agents for example, or forfacilitating controlled release. Depending on their size, the capsulesare millicapsules, microcapsules or nanocapsules, microcapsules beingparticularly preferred for enzymes. Microcapsules are disclosed, forexample, in patent applications WO 97/24177 and DE 19918267. In anotherpossible encapsulation process which starts out from a mixture of theenzyme solution with a solution or suspension of starch or a starchderivative, the enzymes suitable for use in detergents or cleaners areencapsulated in starch or the starch derivative. One such encapsulationprocess is described in German application DE 19956382, “A process forthe production of microencapsulated enzymes”.

Another method of producing a solid composition according to theinvention is tableting or compacting. The tablets produced may besingle-phase or multi-phase tablets. Accordingly, this supply form alsoenables a solid composition according to the invention to be producedwith two solid phases. To produce compositions according to theinvention in the form of single-phase or multi-phase, single-color ormulti-color tablets which may consist of several layers, all theconstituents—optionally per layer—may be mixed together in a mixer andthe resulting mixture may be tabletted in conventional tablet presses,for example eccentric presses or rotary presses, under applied pressuresof about 50 to 100 kN·cm² and preferably 60 to 70 kN/cm². Withmultilayer tablets in particular, it can be of advantage for at leastone layer to be pre-compressed. This is preferably done by applyingpressures of 5 to 20 kN/cm² and more particularly 10 to 15 kN/cm². Atablet produced in this way preferably has a weight of 10 g to 50 g andmore particularly 15 g to 40 g. The tablets may be of any shape,including round, oval or angular and variations thereof.

The enzymes and even a protein essential to the invention—starting froma conventionally performed protein isolation and preparation inconcentrated aqueous or nonaqueous solution—may be added to liquid,gel-form or paste-form compositions according to the invention, forexample, in liquid form, i.e. as a solution, suspension or emulsion, butalso in gel form or in encapsulated form or as a dried powder. Suchdetergents or cleaners according to the invention in the form ofsolutions in typical solvents are generally prepared by simple mixing ofthe ingredients which may be introduced into an automatic mixer as suchor in the form of a solution.

Embodiments of the invention are liquid, gel-form or paste-formcompositions to which a protein essential to the invention and/or one ofthe other proteins present and/or one of the other ingredients presenthave been added in the form of microcapsules. Corresponding compositionscontaining capsules of amylase-sensitive material are particularlypreferred. The use of amylase-sensitive materials and the amylolyticenzyme essential to the invention together in a detergent or cleaner canhave synergistic effects, for example such that the starch-splittingenzyme supports the splitting of microcapsules and hence the release ofthe encapsulated ingredients so that they are only released at a certaintime and not during storage and/or not at the beginning of the cleaningprocess. This mechanism can form the basis of complex detergent orcleaning systems containing various ingredients and various types ofcapsules which represent preferred embodiments of the present invention.

A comparable effect is observed when the ingredients of the detergent orcleaner are distributed among at least two different phases, for exampletwo or more solid, joined-together phases of a tablet-form detergent orcleaner or various granules within the same powder-formdetergent/cleaner. Two-phase or multi-phase cleaners for use both indishwashers and in detergents are already known. The activity of anamylolytic enzyme in a previously activated phase is essential for theactivation of a later phase where it is surrounded by anamylase-sensitive membrane or coating or the amylase sensitive materialis an integral part of the solid phase during whose partial or completehydrolysis the phase in question disintegrates. Accordingly, the use ofthe enzyme essential to the invention for this purpose is a preferredembodiment of the present invention.

The ingredients of detergents/cleaners are able suitably to support oneanother in their performance. The synergistic use of amylase and dyetransfer inhibitors for increasing cleaning performance is disclosed,for example, in patent application WO 99/63035. It is also known thatpolymers which can simultaneously be used as co-builders, such as alkylpolyglycosides for example, can stabilize and increase the activity andthe stability of enzymes present, cf. patent application WO 98/45396. Anamylolytic activity essential to the invention can also be modified,more particularly stabilized and/or increased, by one of the otherconstituents mentioned above. Accordingly, correspondingly adaptedformulations for compositions according to the invention representparticularly preferred embodiments of the present invention. Thisapplies in particular when the washing or cleaning performance of thecomposition is increased in this way.

The basic concept of the present invention is also embodied in processesfor cleaning textiles or hard surfaces which are characterized in thatan amylolytic protein or derivative according to the invention becomesactive in at least one of the process steps.

Machine cleaning processes are distinguished by multistage cleaningprograms, i.e. by the fact that various components with cleaningactivity are applied to the articles to be cleaned at different timesfrom one another. Such processes are applied, for example, in thecleaning of institutional food preparation units. On the other hand,proteins essential to the invention, by virtue of their enzymaticactivity, are themselves capable of attacking carbohydrate-containingsoils, even in the partial or complete absence of detergents or otheringredients characteristic of detergents or cleaners. Accordingly, inone embodiment of the present invention, a machine cleaning process fortextiles or hard surfaces may also be selected in which a proteinessential to the invention acts on the soils for a certain periodwithout other cleaning-active components.

Preferred embodiments of the present invention are any cleaningprocesses, including manual, but especially machine cleaning processes,which are characterized in that an amylolytic protein or derivativeessential to the invention becomes active in at least one of the processsteps. Such processes may be both process for cleaning textiles orcomparable materials and processes for cleaning hard surfaces.

As shown in the Examples, the α-amylase from Bacillus sp. A 7-7 (DSM12368) essential to the invention is suitable, when incorporated inappropriate detergents or cleaners, both for increasing the washingperformance of machine detergents for textiles and for increasing thecleaning performance of dishwasher detergents.

Accordingly, other preferred embodiments of the present invention areany cleaning processes, including manual, but especially machinecleaning processes, which are characterized in that a compositionaccording to the invention is used in at least one of the process steps.Such processes may be both processes for cleaning textiles or comparablematerials and processes for cleaning hard surfaces.

Quantities of protein or fragment according to the inventionparticularly suitable for use in cleaning processes, for example inconventional domestic dishwashers or domestic washing machines, are 0.02mg to 400 mg of the amylolytic protein or fragment, preferably 0.01 mgto 200 mg and more particularly 0.02 mg to 100 mg of the amylolyticenzyme or fragment per application.

In one possible embodiment of machine cleaning processes for textiles orhard surfaces, active concentrations of 0.0005 mg to 10 mg per literwash liquid and preferably 0.005 mg to 8 mg per liter wash liquor haveproved to be particularly suitable for a protein essential to theinvention. Accordingly, they represent preferred embodiments of thepresent invention. In other suitable embodiments, the correspondingvalues may differ significantly from the above figures, particularlywhen it is taken into account that machines consuming between 5 and 50liters of wash liquor for virtually the same quantity of detergent areused for machine cleaning processes.

The use of a protein according to the invention or a compositionaccording to the invention represents another embodiment of the presentinvention. This use may take place by machine or in any other, moreparticularly manual, way. This concerns the cleaning of all kinds ofmaterials, more particularly textiles or hard surfaces. The proteinessential to the invention may be embedded in a formulation of otherdetersive substances or, depending on its nature, may even be largelyunaccompanied by such compounds.

Another corresponding embodiment is the use of a protein according tothe invention on its own for cleaning textiles or hard surfaces, moreparticularly in a multistage cleaning process.

Preferred embodiments are any potential applications where compositionsaccording to the invention for cleaning textiles or hard surfaces areused.

An important use is characterized in that the amylolytic protein orfragment is used in a quantity of 0.02 mg to 400 mg, preferably 0.01 mgto 200 mg and more particularly 0.02 mg to 100 mg per application in adishwasher or washing machine.

The use of proteins or derivatives according to the invention forcompletely or partly dissolving protective layers around constituents ofsolid detergents or cleaners or disintegrating solid phases withamylase-sensitive materials present in, or surrounding, them is anotherembodiment of the present invention. The same also applies to theembodiments where amylolytic proteins are present in one of these phaseseither on their own or together with at least one other detersivesubstance or an active substance which supports the cleaning effect. Tothis extent, the amylolytic protein is intended to activate the relevantphases or other phases in a detergent or cleaner consisting of more thanone phase. The release of the ingredients to produce a cleaning effectof the ingredients on hard surfaces or a textile-like material isparticularly preferred from this perspective also.

The use of the enzyme according to the invention to achieve the partialor complete dissolution of carbohydrate-containing capsules, moreespecially nanocapsules, microcapsules or millicapsules, in liquid,gel-form or paste-form compositions is an embodiment of the presentinvention of which the significance for the controlled release of theencapsulated ingredients of the compositions has already been discussedin the foregoing. This embodiment is a particularly preferred embodimentof the invention when the ingredients are released to produce a cleaningeffect on a hard surface or a textile-like material.

Another embodiment is the use of a protein or derivative according tothe invention for the treatment of raw materials or intermediateproducts in textile manufacture, more particularly for desizing cotton.

Raw materials and intermediate products for textile production, forexample those based on cotton, are finished with starch during theirproduction and further processing in order to facilitate betterprocessing. This process, which is applied both to yarns andintermediate products and to textiles, is known as sizing. Amylolyticproteins according to the invention are suitable for removing the size,i.e. the starch-containing protective layer (desizing).

Another embodiment of the invention are processes for liquefying starch,more particularly for producing ethanol, which are characterized in thatthey use a protein or derivative according to the invention.

For liquefying starch, starch swollen in water or buffer is incubatedwith amylolytic enzymes so that the polysaccharide is split into smallerconstituents, ultimately mainly into maltose. Enzymes according to theinvention are preferably used for such a process or for one of the stepsit involves where their biochemical properties allow them to be readilyintegrated into a corresponding production process. This may the case,for example, when they are to be introduced into a process step inaddition to other enzymes which require the same reaction conditions.Amylolytic proteins according to the invention are particularlypreferred where the products which they themselves form are the focus ofinterest. The liquefaction of starch may also represent one step in amultistage process for producing ethanol or derivatives thereof, forexample acetic acid.

Another embodiment of the invention is the use of a protein orderivative according to the invention for the production of linearand/or short-chain oligosaccharides.

By virtue of their enzymatic activity, amylolytic proteins according tothe invention mainly form relatively high molecular weightoligosaccharides, such as maltohexaose, maltoheptaose or maltooctaosefor example, from starch-like polymers after a relatively shortincubation period. After a relatively long incubation period, there isan increase in the proportion of lower oligosaccharides, such as maltoseor maltotriose for example, among the reaction products. Where there isa particular interest in certain reaction products, correspondingvariants of proteins according to the invention and/or the reactionconditions may be modified accordingly. This is particularly attractivewhen pure compounds are not the main concern, but rather mixtures ofsimilar compounds as, for example, in the formation of solutions,suspensions or gels having only certain physical properties.

Another embodiment is the use of a protein or derivative according tothe invention for the hydrolysis of cyclodextrins.

Cyclodextrins are α-1,4-glycosidic cyclic oligosaccharides among whichthose of 6, 7 or 8 glucose monomers, the α-, β- and γ-cyclodextrins (orcyclohexa-, -hepta- or -octa-amyloses), have the greatest economicimportance. With hydrophobic guest molecules, such as perfumes, flavorsor pharmaceutical active principles for example, they are capable offorming inclusion compounds from which the guest molecules can bereleased as required. Depending on the field of application of theingredients, such inclusion compounds are of importance, for example,for the production of foods, pharmaceutical or cosmetic products, forexample in corresponding products for the end consumer. Accordingly, therelease of ingredients from cyclodextrins is a practical application forproteins according to the invention.

Another embodiment is the use of a protein or derivative according tothe invention for the release of low molecular weight compounds frompolysaccharide carriers or cyclodextrins.

By virtue of their enzymatic activity, amylolytic proteins according tothe invention are also capable of releasing low molecular weightcompounds from other α-1,4-glycosidic polysaccharides. As with thecyclodextrins, this can take place at the molecular level and even onlarger systems such as, for example, ingredients encapsulated in theform of microcapsules. Starch, for example, is an established materialfor encapsulating such compounds as, for example, enzymes—which are tobe introduced into reaction mixtures in defined quantities—duringstorage. The controlled release process from such capsules can besupported by amylolytic enzymes according to the invention.

Another embodiment is the use of a protein or derivative according tothe invention for the production of foods and/or food ingredients.

The use of a protein or derivative for the production of animal feedand/or animal feed ingredients is another embodiment of the presentinvention.

Wherever starch or starch-derived carbohydrates play a role asingredients of foods or animal feeds, an amylolytic activity may be usedfor the production of these articles. It increases the proportion ofmono- or oligomers in relation to the polymeric sugar which can be tothe benefit of the taste, digestibility or consistency of the food. Thismay be necessary for the production of certain animal feeds and also,for example, in the production of fruit juices, wine or other foods ifthe level of polymeric sugars is to be reduced and the level of sweetand/or more readily soluble sugars increased. The above-mentionedapplication for liquefying starch and/or producing ethanol may beregarded as an industrial variant of this principle.

In addition, amylases also counteract the loss of taste from bread andconfectionery through staleness (anti-staling effect). To this end, theyare suitably added to the dough before baking. Accordingly, preferredembodiments of the present invention are those in which proteinsaccording to the invention are used for the production of bread andconfectionery.

Another embodiment is the use of a protein or derivative according tothe invention for dissolving starch-containing adhesive bonds.

Temporary bonding processes which are characterized in that they use aprotein or derivative according to the invention represent anotherembodiment of the present invention

Besides other natural materials, starch has been used for centuries as abinder in paper manufacture and in the bonding of different papers andpaperboards. This applies, for example, to graphics and books. Over longperiods, such papers can become wavy or can break under unfavorableinfluences, such as moisture for example, resulting in their completedestruction. To restore such papers and boards, it may be necessary todissolve the adhesive layers which is made very much easier by the useof an amylolytic protein according to the invention. Vegetable polymers,such as starch or cellulose and water-soluble derivatives thereof, areused as adhesives or pastes. To this end, they first have to be swollenin water and then dried after application to the surface to be glued sothat it is fixed to the substrate. The enzyme according to the inventionmay be added to such an aqueous suspension in order to influence theadhesive properties of the paste formed. However, instead of or inaddition to this function, it may also be added to the paste in order tostay inactive on the surface for a long time after drying, for examplefor a few years. Controlled changes in the ambient conditions, forexample through moistening, can be used to activate the enzyme at alater date and hence to induce dissolving of the paste. In this way, theglued surface is easier to separate from the substrate. In this process,the enzyme according to the invention, through its amylolytic activity,acts as a separating agent in a temporary bonding process or as aso-called “switch” for separating the glued article.

EXAMPLES Example 1

The candidates for a microbiological screening for amylase-formingmicroorganisms with the selection criterion growth and halo formation onagar plates with starch as sole carbon source included the Bacillusstrain Bacillus sp. (RNA group VI, alcaliphilic) A 7-7 which has beenlodged at the DSMZ (DSM ID 98-587, lodgement DSM 12368).

The culture medium used was YPSS medium containing 15 g/l solublestarch, 4 g/l yeast extract, 1 g/l K₂HPO₄ and 0.5 g/l MgSO₄×7 H₂O. Afterautoclaving, the pH was adjusted to 10.3 with 20% sodium carbonatesolution. Quantities of 25 ml medium were introduced into sterile 100 mlErlenmeyer flasks with chicane and inoculated with a culture of Bacillussp. A 7-7 (DSM 12368) which had been grown on a YPSS agar plate.Cultivation was carried out with shaking over 48 h at 30° C./200 r.p.m.

Example 2

A singular enzyme was obtained from the culture medium through thefollowing purification steps: precipitation of the culture supernatantwith ethanol; taking up the protein pellet in 50 mM tris/HCl buffer, pH8.5; dialysis against 50 mM tris/HCl buffer, pH 8.5; cation exchangechromatography on Fast-flow-S-Sepharose® (Pharmacia-Amersham Biotech,Freiburg) with 50 mM tris/HCl buffer, pH 8.5 as eluent; rebuffering ofthe amylase-containing break-through against 20 mM glycine/NaOH buffer,pH 10, in PD10® columns (Pharmacia-Amersham Biotech); anion exchangechromatography on Mono-Q®(Pharmacia-Amersham Biotech) using the samebuffer as eluent with an increasing salt gradient of 0 to 1 M NaCl. Theamylase eluted at ca. 0.25 M NaCl.

2.6 mg of a protein were obtained in this way from 250 ml of culturemedium, as determined by the BCA method (bicinchonic acid;2,2′-biquinolyl-4,4′-dicarboxylic acid). The protein was shown to bepure by SDS gel electrophoresis and coloring with silver.

In denaturing SDS polyacrylic gel electrophoresis in an 8-25% gel in thePHAST® system (Pharmacia-Amersham) and in comparisons with relevant sizemarkers, the amylolytic enzyme from Bacillus sp. A 7-7 (DSM 12368) has amolecular weight of 58 kD.

According to isoelectric focussing from pH 3 to 9 in the PHAST® system(Pharmacia-Amersham), its isoelectric point lies at 6.0.

The amylolytic activity of the purified enzyme was determined by theso-called DNS method, i.e. using dinitrosalicylic acid. To this end, theoligosaccharides disaccharides and glucose units released by the enzymein the hydrolysis of starch are detected by oxidation of the reducingends with dinitrosalicylic acid (DNS). The intensity of colordevelopment is proportional to the number of cleavage products formed.This test is carried out as follows: after incubation of 50 μl enzymesolution with 100 μl 1% soluble starch in tris/maleate buffer, pH 6.5(12.11 g tris+11.61 g maleic acid to 1 liter, pH adjusted with 0.2 NNaOH) for 15 mins. at 50° C., the reduced saccharides are oxidized for20 mins. at 100° C. with 300 μl DNS solution (8.8 g dinitrosalicylicacid, 915 ml distilled water, 250 g K—Na tartrate, 334 ml 4.5% NaOH, 6.3g sodium disulfite). After cooling in an ice bath, absorption isphotometrically detected at 540 nm against a blank value. The assay iscalibrated via a maltose concentration series. The activity is expressedin μmol reducing sugars (based on maltose) per min. and ml.

According to this test, the protein of the Examples clearly hasamylolytic activity. In the following, the activity determined in thisway serves as a parameter for the stability of the enzyme under variousconditions.

The temperature stability of the enzyme was measured in 10-minuteincubations at a pH of 10. At room temperature, 30° C., 40° C. and 50°C., the activity is at least 85%. At 40° C., the amylase has its maximumof 90% residual activity. At 60° C., the enzyme has 50% activity. Attemperatures above 60° C., it undergoes a serious loss of activity, butstill has 10% residual activity at 80-90° C.

The amylolytic enzyme from Bacillus sp. A 7-7 (DSM 12368) is largelystable at pH values of 5 to 12 when incubated for 10 mins. at 40° C. AtpH values of 8 to 9, the amylase is up to 100% stable. At higher andlower pH values, its activity slowly decreases although the enzyme stillhas more than 65% residual activity at pH 5 and pH 12.

For further characterization, the adverse effect on enzymatic activityof possibly disturbing factors, such as protease or detergents, isinvestigated. After exposure to 10 HPE/ml of the protease Savinase®(Novo Nordisk A/S, Bagsvaerd, Denmark; the HPE units can be determinedby the method reported in Tenside 7 (1970), pp. 125-132) and 0.1% SDSfor 15 minutes at pH 10/50° C., the enzyme shows 74% residual activity.After exposure to 0.1% SDS under the same conditions (pH 10, 15 minutes,50° C.), the enzyme has 98% residual activity. Still under theseconditions, it has 10% residual activity in the presence of 3 mM EDTAand 65% residual activity in the presence of 1 mM EDTA.

Example 3

All molecular-biological process steps follow standard methods which aredescribed, for example, in manuals such as the manual by Fritsch,Sambrook and Maniatis entitled “Molecular cloning: a laboratory manual”,Cold Spring Harbour Laboratory Press, New York, 1989.

In order to determine internal amino acid sequences, the proteinobtained in accordance with Example 1 and purified in accordance withExample 2 was first precipitated with ethanol and then separated bydenaturing SDS polyacrylamide gel electrophoresis. After thecorresponding bands had been cut out from the SDS polyacrylamide gel,digestion was carried out tryptically in situ, the resulting peptideswere separated by HPLC and analyzed by Edman degradation and MALDI-TOFanalysis. The following two fragments D1 (SEQ ID NO: 6) and D6 (SEQ IDNO: 8) were selected from the many tryptic fragments obtained in thisway:

D1: GITAVWIPPAWK (SEQ ID NO: 6) 5′-GTN TGG ATH CCN CCN GCN TGG-3′ (SEQID NO: 7) D6: QGYPSVFYGDYYGIPTH (SEQ ID NO: 8) 5′-GGD ATN CCN TAN TARTCN CC-3′ (SEQ ID NO: 9)

The corresponding degenerated PCR primers were constructed from theiramino acid sequences (shown on the left). Their nucleotide sequences areshown on the right (N stands for any base; H stands for A or C or T; Rstands for A or G; D stands for A, G or T).

From normally prepared chromosomal DNA of Bacillus sp. A 7-7 (DSM 12368)as matrix, a ca. 1,000 bp large fragment was amplified with this primerpair in a standard PCR. For storage, this PCR fragment was cloned in thevector pGEM-Teasy® (Promega, Madison, Wis., USA).

Example 4

Using the nonradioactive DIG-High-Prime® labeling kit of BoehringerMannheim (Germany: product No. 1245832), the purified PCR fragment waslabeled as directed as a DNA probe. For subsequent hybridization,chromosomal DNA of the strain Bacillus sp. A 7-7 (DSM 12368) was splitwith the restriction enzyme Xba 1 and separated via a 0.9% agarose gel.The DNA was transferred by a vacuum blotter to a positively chargednylon membrane (Roche, Mannheim). DNA hybridization and immunologicaldetection of the PCR fragment used as probe were carried out accordingto the directions of the DIG-High-Prime® Labeling-and-Detection StarterKits I® of Boehringer Mannheim. It was found that the target gene ispresent in an apparently ca. 3,000-4,000 large DNA section. Thefragments of this size were purified via a 0.9% agarose gel and thenligated in the vector pCB56C (from B. Subtilis DB 104; described inapplication WO 91/02792) cut with the restriction enzymes Xba I and NheI. After transformation in an amylase-negative Bacillus subtilis strain,amylase-positive clones were identified through halo formation onstarch-containing agar plates. A representative isolate carried therequired insert.

Example 5

The sequencing of the plasmid obtained was carried out by standard chaintermination methods. The plasmid contained an insert with a size of2,015 bp. On top lies the 1,545 bp large gene for the interesting enzymewhich is shown under SEQ ID NO. 1 in the sequence protocol of thepresent application. To this corresponds a polypeptide of 516 aminoacids of which the sequence is shown in SEQ ID NO. 2 in the sequenceprotocol. The molecular weight derived from this amino acid sequence is59 kD or—for the mature protein obtained by splitting off the signalpeptide with 31 amino acids—55.5 kD.

Sequence comparisons by the BLAST method (S. F. Altschul et al., Nucl.Acids Res., 25 (1997), pp. 3389-3402) carried out on Mar. 17, 2000characterize the enzyme as α-amylase. At the protein level, the homologyof this protein to known proteins is between 71 and 95% identity, asshown in Table 2 below.

TABLE 2 Homology of the enzyme according to the invention from Bacillussp. A 7-7 (DSM 12368) to the most similar and other representativeproteins; expressed in % identity at protein level. Organism ID Identity[%] B. alcalophilus P 19571 95 B. alcalophilus WO 96/23873 91 B.licheniformis P 06278 72 B. amyloliquefaciens P 00692 72 B.stearothermophilus P 06297 71 ID stands for the entries at the SwissProt Data Bank (Geneva, Switzerland).

An alignment with representative proteins is shown in FIG. 1. All theproteins are α-amylases so that, on the basis of the substantialaccords, it must be assumed that the enzyme according to the inventionis also an α-amylase. This is in line with the original isolation of thegene from a starch-degrading microorganism (Example 1) and the discoveryof the amylolytic activity of a transformant with the insert-carryingplasmid (Example 4). The properties of the amylolytic protein obtainablefrom this production strain correspond with those of the wild typestrain (Example 2). Such a production strain can be produced inaccordance with Example 4.

Example 6

Cotton fabrics were treated under standardized conditions with the fourdifferent soils A (chocolate mousse), B (oat flakes with cocoa), C (oatflakes with cocoa and a little milk) and D (potato starch) and, usingthe material thus prepared, various detergent formulations werelaunderometer-tested for washing performance. For the tests, a liquorratio of 1:12 was adjusted and the fabrics were washed for 30 mins. at30° C. The dosage was 5.88 g of the particular detergent per liter washliquor. The water hardness was 16° German hardness.

The control detergent for A, B and C was a basic detergent with thefollowing composition (in % by weight): 4% linear alkyl benzenesulfonate(sodium salt), 4% C₁₂₋₁₈ fatty alcohol sulfate (sodium salt), 5.5%C₁₂₋₁₈ fatty alcohol×7 EO, 1% sodium soap, 11% sodium carbonate, 2.5%amorphous sodium disilicate, 20% sodium perborate tetrahydrate, 5.5%TAED, 26% zeolite A, 4.5% polycarboxylate, 0.5% phosphonate, 2.5%granular foam inhibitor, 5% sodium sulfate, 1% protease granules, rest:water, optical brightener, perfume, salts. For the various test series,various amylases were added to the control detergent so that the finalconcentration was 44 TAU of amylolytic activity per liter wash liquor.

The amylolytic activity in TAU was determined using a modifiedp-nitrophenyl maltoheptaoside of which the terminal glucose unit isblocked by a benzylidene group which is split by amylase to freep-nitrophenyl oligosaccharide which in turn is reacted with theauxiliary enzymes glucoamylase and alpha-glucosidase to form glucose andp-nitrophenol. The quantity of p-nitrophenol released is thusproportional to the amylase activity. The measurement is carried out,for example, with an Abott Quick-Start® Testkit (Abott Park, Ill., USA).The increase in absorption (405 nm) in the test mixture is detected over3 mins. at 37° C. against a blank value using a photometer. An enzymestandard of known activity is used for calibration (for exampleMaxamyl®/Purastar® of Genencor with an activity of 2,900 TAU/g).Evaluation is carried out by plotting the absorption difference dE (40 5nm) per min. against the enzyme concentration of the standard.

The amylolytic enzyme according to the invention from Bacillus sp. A 7-7(DSM 12368) was compared with Termamyl®, Duramyl® and BAN®(manufacturer: Novo Nordisk A/S, Bagsvaerd, Denmark). The detergent usedfor soil D was the same basic formulation, but without protease, used ascontrol for A-C or with added amylases.

In the present Example, the whiteness of the fabrics in the CIELABsystem was measured before and after washing with a Minolta CR 310 incomparison with a standard which was standardized to 100%. Thedifferences in the results obtained are set out in Table 3 below for therespective tests. The average values of 5 measurements are shown. Theyare a direct indication of the contribution of the enzyme present to thewashing performance of the detergent used.

TABLE 3 Basic detergent containing A B C D Amylase essential to theinvention 29.3 26.0 20.9 15.2 Termamyl ® 25.9 22.7 17.4 12.3 Duramyl ®28.6 23.4 20.2 14.2 BAN ® 22.5 22.0 17.0 13.2 Control without amylase22.9 22.2 11.9 10.0 Standard deviation 1.3 0.6 1.4 2.2

It can be seen that the α-amylase from Bacillus sp. A 7-7 (DSM 12368)produces distinctly better washing performances against soil B than anyof the three reference enzymes. Against the other soils, it showsslightly improved washing performance (within the limit of error)although this is still well above the comparison values withoutamylolytic enzyme. This result is all the more conclusive insofar as allthe detergent formulations tested contain a bleaching agent to whichwild type enzymes are generally very sensitive.

Example 7

Cotton fabrics were treated under standardized conditions with soils B(oat flakes with cocoa) and C (oat flakes with cocoa and a little milk).Launderometer testing was carried out as in Example 1 using anotherdetergent formulation, namely (in % by weight): 14% Na alkylbenzenesulfonate, 6% Na fatty alcohol sulfate, 6% 7×-ethoxylated C₁₂₋₁₈fatty alcohol, 1% soap, 25% zeolite NaA, 10% Na carbonate, 5% polymericpolycarboxylate (Sokalan® CP5), 11% trisodium citrate dihydrate, 4%citric acid, 1% particulate foam inhibitor, 1% protease granules, 5%sodium sulfate, rest: water and salts. For the various test series,various amylases were added to this basic formation so that the finalconcentration was 33.5 TAU of amylolytic activity per liter of washliquor, as determined by the method described in Example 1. Theamylolytic enzyme essential to the invention from Bacillus sp. A 7-7(DSM 12368) was compared with Termamyl®, Duramyl® and BAN®(manufacturer: Novo Nordisk A/S, Bagsvaerd, Denmark). The dosage was4.45 g of the particular detergent per liter wash liquor.

After washing, the whiteness of the washed fabrics was determined in thesame way as in the preceding Example. The differences obtained are setout in Table 4 and represent the average values of 5 measurements which,again, are a direct indication of the contribution of the particularenzyme to the washing performance of the detergent.

TABLE 4 Basic detergent containing B C Amylase essential to theinvention 30.6 17.5 Termamyl ® 29.3 15.0 Duramyl ® 29.2 16.7 BAN ® 28.915.6 Control without amylase 28.5 14.5 Standard deviation 0.6 1.2

It can be seen that the α-amylase from Bacillus sp. A 7-7 (DSM 12368) inthis bleach-free detergent formulation produces better washingperformance against soils B and C than any of the reference enzymes.

Example 8

Cotton fabrics were soiled under standardized conditions with twodifferent types of commercially available milk cocoa (E and F).Launderometer testing was then carried out as described in Example 1.The control detergent used was the basic detergent formulation ofExample 2, but without protease. For the various test series, thevarious amylases were added in the same way as in Example 2. Thedetergent was used in the same dosage.

After washing, the whiteness of the washed fabrics was measured incomparison with that of barium sulfate which was standardized to 100%.The measurement was carried out using a Datacolor SF500-2 spectrometerat 460 nm (UV blocking filter), 30 mm orifice, without gloss, light typeD65, 10°, d/8°. The results obtained are set out as % reflectance, i.e.as percentages in comparison with barium sulfate, in Table 4 below whichalso shows the particular starting value. The values shown are theaverages of 5 measurements and are a direct indication of thecontribution of the amylolytic enzyme present to the washing performanceof the detergent used.

TABLE 5 Basic detergent containing E F Amylase essential to theinvention 70.3 41.0 Termamyl ® 67.3 39.7 Duramyl ® 68.3 40.5 BAN ® 68.739.8 Control without amylase 61.1 31.4 Starting value 21.1 25.0 Standarddeviation 1.0 1.2

It can be seen that the α-amylase from Bacillus sp. A 7-7 (DSM 12368) inthis formulation produces distinctly better washing performance in thecase of E than any of the reference systems tested and, in the case ofF, comparable washing performance.

Example 9

For application-oriented cleaning tests, vessels with hard smoothsurfaces were provided with the following soils under standardizedconditions: A (DIN oat flakes), B (oat flakes swollen in water) and C(mixed starch), and were washed at 45° C. with the normal program of adomestic dishwasher (Miele® G 575). The detergent dosage was 20 g percleaning cycle and the water hardness was 16° German hardness.

The following basic formulation was used as the detergent (allquantities in % by weight): 55% sodium tripolyphosphate (expressed aswater-free), 4% amorphous sodium disilicate (expressed as water-free),22% sodium carbonate, 9% sodium perborate, 2% TAED, 2% nonionicsurfactant, 1.4% protease granules, rest: water, dyes, perfume. For thevarious tests, various amylases, namely Termamyl®, Duramyl®, BAN®(manufacturer: Novo Nordisk A/S, Bagsvaerd, Denmark), or the amylolyticenzyme from Bacillus sp. A 7-7 (DSM 12368) were added to the basicformulation in effective quantities of 150 TAU of amylolytic activityper cleaning cycle, as determined by the method described in Example 1.

After washing, the removal of soil A was evaluated after coloring withiodine by the iodine/starch reaction, evaluation being carried outvisually on a scale of 0 (=unchanged, i.e. very pronounced soil) to 10(=no soil discernible). The removal of soils B and C was determinedgravimetrically in %. To this end, the difference between the weight ofthe soiled and then cleaned vessel and the starting weight of the vesselwas related to the difference in weight between the uncleaned vessel andits starting weight. This relation may be regarded as percentageremoval.

The results obtained are set out in Table 6 below where they are shownas the averages of 9 measurements for A and B and 18 measurements for C.They are a direct indication of the contribution of the enzyme presentto the cleaning performance of the detergent used.

TABLE 6 B C Basic detergent containing A % removal % removal Amylaseessential to the invention 6.2 91.2 95.8 Termamyl ® 3.7 53.9 15.7Duramyl ® 2.2 78.8 35.5 BAN ® 3.7 69.1 34.2 Control without amylase 4.331.7 0.6

These results show that the α-amylase from Bacillus sp. A 7-7 (DSM12368) is superior to all the other amylases tested in its cleaningperformance against all three soils in dishwasher detergents at 45° C.

Example 10

Vessels with hard smooth surfaces were provided with the same soils asin the preceding Example and washed at 55° C. The cleaning conditionsand the formulation of the detergents used also corresponded to those ofthe preceding Example.

After washing, the removal of soil A was visually evaluated as inExample 4 on a scale of 0 to 10 using the iodine/starch reaction. Theremoval of soils B and C was determined gravimetrically in %, again asin Example 4. The results obtained are set out in Table 6 below wherethey are shown as the averages of 9 measurements for A, 10 measurementsfor B and 18 measurements for C. They are a direct indication of thecontribution of the enzyme present to the cleaning performance of thedetergent used.

TABLE 6 B C Basic detergent containing A % removal % removal Amylaseessential to the invention 8.3 97 99 Termamyl ® 6.7 95 53 Duramyl ® 6.995 89 BAN ® 6.2 92 77 Control without amylase 5.3 71 27

These results show that the α-amylase from Bacillus sp. A 7-7 (DSM12368) is superior to all the other amylases tested in its cleaningperformance against all three soils in dishwasher detergents, even at adishwashing temperature of 55° C.

DESCRIPTION OF THE FIGURES

FIG. 1:

Alignment of the amylolytic enzyme according to the invention fromBacillus sp. A 7-7 (DSM 12368) with the three most similar amylases.

In FIG. 1,

-   -   1=amylase from Bacillus sp. A7-7 (DSM 12368) (SEQ ID NO: 2)    -   2=mature amylase from Bacillus sp. # 707 according to:        Tsukamoto, Kimura, Ishii, Takano and Yamane, Biochem. Biophys.        Res. Common. 151 (1988), pp. 25-31 (SEQ ID NO: 3)    -   3=amylase of SEQ ID NO. 1 from WO 96/23873 (SEQ ID NO: 4)    -   4=amylase of SEQ ID NO. 2 from WO 96/23873 (SEQ ID NO: 5)

1. Bacteria characterized as a member of the genus Bacillus sp. A 7-7(DSM 12368) and from which is isolated a polypeptide having amylolyticactivity and comprising an amino acid sequence that is selected from thegroup consisting of a sequence that is at least 96% identical to SEQ IDNO:2 and a sequence that is at least 96% identical to residues 32 to 516of SEQ ID NO:2.